Purification and properties of rabbit uterus preuteroglobin mRNA

Amemann, Jutta; Heins, Bärbel; Beato, Miguel

doi:10.1093/nar/4.11.4023

Cited by 21 publications

(19 citation statements)

References 26 publications

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“…Injection of the rabbits, preparation of the endometrium and isolation of total polysomal RNA were as previously reported [2,3]. Fractionation of endometrial polysomes into the free and membrane-bound fraction was performed by a modification of the procedure of Marcu et al [4].…”

Section: Purification Of M R N Amentioning

confidence: 99%

“…The poly(A)-containing RNA was purified from polysomal RNA by repeated (twice) chromatography on oligo(dT)-cellulose, and centrifuged through sucrose density gradients as previously reported [2]. Before each step the RNA was heated at 65°C and low salt for 5 min.…”

Section: Purification Of M R N Amentioning

confidence: 99%

“…Before each step the RNA was heated at 65°C and low salt for 5 min. The pooled fractions from the sucrose gradients were precipitated with ethanol, redissolved and electrophoresed in 4 % polyacrylamide gels prepared in purified formamide as described [2,5]. After scanning at 270 nm, the desired bands were cut, and passed three times through a 20-gauge needle in 1 ml of extracting medium (0.6 M LiCl, 20 mM sodium acetate, pH 7.5, 0.5% sodium dodecylsulfate and 1 mM Na2EDTA).…”

Section: Purification Of M R N Amentioning

confidence: 99%

“…Electrophoresis was carried out in 5 % polyacrylamide gels containing 90 mM Tris/borate (pH 8.3) and 2 mM EDTA [6]. Fluorography was performed as described [2].…”

Section: Purification Of M R N Amentioning

confidence: 99%

“…In a recent paper we have reported the partial purification of the mRNA for pre-uteroglobin which can be used as a template for the synthesis of a complementary DNA [2]. This complementary DNA is needed as a probe for the quantification of specific pre-uteroglobin mRNA sequences during hormonal induction, and could also serve to synthesize a double-stranded DNA fragment suitable for DNA recombination experiments.…”

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Synthesis and Characterization of a DNA Complementary to Pre‐uteroglobin mRNA

Arnemann

Heins

Beato

1979

European Journal of Biochemistry

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Uteroglobin, a progesterone-induced uterine protein of the rabbit, is synthesized in cell-free systems as a precursor containing 21 additional amino-acids at its N-terminal end. The mRNA for pre-uteroglobin has been purified from the membrane-bound polysomes of induced endometrium and used as template for the synthesis of a full copy complementary DNA. Final purification of the cDNA was based on hybridization to the template mRNA up to a low value of rot (0.01 M . s) and digestion of the non-hybridized cDNA by S1 nuclease.A comparison of the hybridization kinetics of the pre-uteroglobin cDNA and rabbit globin cDNA to their respective templates indicates a nucleotide sequence complexity of 650 for pre-uteroglobin mRNA, in agreement with the values obtained by sucrose gradient centrifugation and polyacrylamide gel electrophoresis in formamide. The melting temperature of the hybrids of pre-uteroglobin cDNA to its template reflects the absence of mismatched sequences. This cDNA has been used to quantify pre-uteroglobin mRNA sequences in the endometrial RNA from control animals and from animals treated sequentially with estradiol and progesterone. In agreement with the induction of uteroglobinsynthesizing activity, there is a dramatic increase in the uterine content of pre-uteroglobin mRNA after hormonal treatment. Part of this effect can be accounted for by hormonally induced cell proliferation. When expressed on a DNA basis there is a 50-100-fold increase in the cellular content of pre-uteroglobin mRNA following hormonal treatment.The synthesis of uteroglobin in rabbit endometrium is regulated by ovarian steroids, and its induction offers a suitable model system for studying the molecular mechanism of steroid hormone action on specific protein synthesis (for review see [I 3 ) . In a recent paper we have reported the partial purification of the mRNA for pre-uteroglobin which can be used as a template for the synthesis of a complementary DNA [2]. This complementary DNA is needed as a probe for the quantification of specific pre-uteroglobin mRNA sequences during hormonal induction, and could also serve to synthesize a double-stranded DNA fragment suitable for DNA recombination experiments.In this paper we describe an improved purification method for pre-uteroglobin mRNA and present our data on the synthesis, purification, and properties of its cDNA. Using this probe in RNA-excess hydridizaAbbreviations. rot, product o f R N A concentration (in moles of nucleotides per liter) and time (in seconds); r0tli2, rot at which the hybridization of a given population of nucleic acid molecules is half completed.Enzymes. S 1 nuclease or single-stranded nuclease (EC 3.1.4.21); deoxyribonuclease I (EC 3.1.4.5); reverse transcriptase or RNAdirected DNA polymerase (EC 2.7.7.7). tion reactions, we show that there is an accumulation of pre-uteroglobin mRNA sequences in endometrial RNA following hormonal induction of uteroglobin synthesis. MATERIALS AND METHODS Materials ~-[~~S]Methionine (600 Ci/mmol) and [3H JdCTP (22 Ci/mmol) were ob...

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Section: Purification Of M R N Amentioning

confidence: 99%

Section: Purification Of M R N Amentioning

confidence: 99%

Section: Purification Of M R N Amentioning

confidence: 99%

“…Electrophoresis was carried out in 5 % polyacrylamide gels containing 90 mM Tris/borate (pH 8.3) and 2 mM EDTA [6]. Fluorography was performed as described [2].…”

Section: Purification Of M R N Amentioning

confidence: 99%

mentioning

confidence: 99%

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Synthesis and Characterization of a DNA Complementary to Pre‐uteroglobin mRNA

Arnemann

Heins

Beato

1979

European Journal of Biochemistry

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Regulation of the Expression of the Uteroglobin Gene by Ovarian Hormones

Beato

Arnemann

Menne

et al. 1983

Regulation of Gene Expression by Hormones

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Ultrastructural and kinetic studies on uteroglobin secretion in the uterus and oviduct of the pseudopregnant rabbit

1987

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The intracellular localization of uteroglobin, a progesterone-induced protein, was studied in uterus and oviduct by means of immunoelectron microscopy with the protein A-gold technique. In the uterus, uteroglobin was synthesized in the columnar epithelium of the endometrium where most of the cells were immunoreactive. The protein was localized mainly in small secretory granules which were seen in the process of release into the uterine lumen. The luminal microvilli were also heavily stained. In the oviduct, the secretory cells contained large immunoreactive granules at the apical zone, some of which were observed while discharging into the lumen. Within these secretory granules, uteroglobin accumulated particularly in lens-shaped patches at the periphery of the granules. In vitro kinetic studies on the secretion of newly synthesized uteroglobin indicated that the ability to store uteroglobin is greater in the oviduct than in the uterus; however, the rate of uteroglobin secretion is greater in the uterus than in the oviduct. Thus, there appears to be a good correlation between the microscopic and the functional observations.

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Purification and properties of rabbit uterus preuteroglobin mRNA

Cited by 21 publications

References 26 publications

Synthesis and Characterization of a DNA Complementary to Pre‐uteroglobin mRNA

Synthesis and Characterization of a DNA Complementary to Pre‐uteroglobin mRNA

Regulation of the Expression of the Uteroglobin Gene by Ovarian Hormones

Ultrastructural and kinetic studies on uteroglobin secretion in the uterus and oviduct of the pseudopregnant rabbit

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