NaCl/Cit, 0.15 M sodium chloride, 0.015 M trisodium citrate,. pH 7.0. ... A lesser degree of variation was ... NaCl/Cit at 42°C and finally with 0.1 x NaCljCit at.
Eur. J. Biochem. 112, 443-450 (1980) by FEBS 1980
Characterization of Cloned Complementary DNA Covering more than 6000 Nucleotides (97 %) of Avian Vitellogenin mRNA Peter J. COZENS, Andrew C. B. CATO, and Jean-Pierre JOST Friedrich-Miescher-Institut, Basel
(Received June 30, 1980)
The messenger RNA coding for chicken vitellogenin, a precursor of the egg-yolk proteins lipovitellin and phosvitin, is synthesized in the liver following estrogen injection. This mRNA is 6600 nucleotides long. We have previously reported the cloning and preliminary characterization of some cDNA fragments representing portions of the vitellogenin mRNA [Biochem. Biophys. Acta, 606, 34-46 (1980)l. In this paper we report the full characterization of a larger series of such clones, representing almost the entire length of the mRNA, by restriction endonuclease mapping, R-loop mapping, RNA-DNA hybridization and by translation in vitro of the RNA which hybridizes to the cloned DNA, From the results we conclude that the chicken vitellogenin mRNA, unlike that of Xenopus luevis, does not vary in sequence over most of its length, although some variations in the cDNA sequences were detected particularly in clones derived from the 3' terminus of the RNA. All sequence variants appearto be present in RNA prepared from single animals. The possible origins of these minor species are discussed. Furthermore, we describe a cDNA clone complementary to an mRNA which is about the same size as vitellogenin mRNA and which codes for an egg yolk protein antigenically related to lipovitellin. This mRNA is synthesized constitutively.
Vitellogenin is a protein with a monomeric molecular weight of about 220000 which is synthesised in the livers of egg-laying vertebrates (both male and female) in response to treatment with estrogen. It is secreted into the blood stream and transported to the oocytes where it is cleaved to form the yolk proteins, lipovitellin and phosvitin (for recent reviews see [1,2]). The induction of vitellogenin synthesis in the avian liver by 17P-estradiol is a particularly advantageous system for the investigation of gene regulation since the hormone induces large amounts of mRNA in a homogenous cell population. Moreover, inhibition of DNA synthesis does not impair vitellogenin induction, implying that cell proliferation is not needed [3]. Experiments using cDNA-mRNA hybridization have indicated that the numbers of vitellogenin mRNA molecules increase dramatically from an undetectable level to about 7000 molecules Abbreviations. pre-mRNA, precursor to messenger RNA; NaCl/Cit, 0.15 M sodium chloride, 0.015 M trisodium citrate, pH 7.0. Enzymes. Restriction endonucleases: A m 1 (EC 3.1.23.3); Bull (EC 3.1.23.5); BumHI (EC 3.1.23.6); Bgfl (EC 3.1.23.9); Hue111 (EC 3.1.23.17); HhuI (EC 3.1..19); Hind11 (EC 3.1.23.20); Hind111 (EC 3.1.23.21); Hinfl (EC 3.1.23.22); KpnI (EC 3.1.23.26); MboII (EC 3.1.23.28); PstI (EC3.1.23.31); PzuI (EC3.1.23.31); Puul (EC 3.1.23.32); PuuII (EC 3.1.23.33); Sac1 (EC 3.1.23.24); SulI (EC 3.1.23.37); TuyI(EC 3.1.23.39).
per cell during primary stimulation with estrogen [4,5]. This strongly indicates that estrogen increases the level of transcription of the vitellogenin genes. We have previously shown that vitellogenin premRNA is about twice the size of the mature mRNA [6], which suggests that the primary gene transcript contains non-coding sequences. We are currently interested in the problem of the structural organization of the avian vitellogenin gene and its pre-mRNA and in the effects of estrogen on this gene at the transcriptional (and post-transcriptional) level. To investigate these questions, it is necessary to isolate both the vitellogenin gene from cellular DNA and vitellogenin cDNA in large amounts with absolute sequence purity. This can only be done by cloning these DNA species. This report describes an extensive characterization of a large number of cloned vitellogenin cDNA species by restriction mapping, R-loop mapping and DNA-RNA hybridisation. Owing to the large size of vitellogenin mRNA (about 6600 nucleotides [7,8]) we were unable to obtain a single cDNA species which covered the entire vitellogenin mRNA. However, we did obtain a series of four overlapping cDNA sequences which together represent all regions of the vitellogenin message. The identification of these four clones involved analysis of a large number of vitello-
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Characterization of Cloned Avian Vitellogenin cDNA
genin cDNA sequences. This analysis showed that there is extensive sequence variation at the 3' end of vitellogenin mRNA. A lesser degree of variation was detected elsewhere on the molecule. All these variants appear to be expressed in a single animal. The significance of these variations and their relationship to the subgroups of vitellogenin mRNA sequences found in Xenopus [9] is discussed. Furthermore, this report describes the characterization of a vitellogenin cDNA sequence which was synthesized from an mRNA species that is apparently produced constitutively. MATERIALS AND METHODS Restriction Mapping of Recombinant Plasm ids
All restriction endonucleases were purchased from New England Biolabs and digestions with single enzymes were carried out in the buffers recommended by the supplier. The conditions for simultaneous digestion with several enzymes were determined empirically. Digested fragments were analysed by electrophoresis on vertical slab gels (20-cm long by 4-mm thick) using an applied voltage of less than 2 V/cm. The sizes of fragments having less than 600 base pairs were determined on 5 "/, or 8 % polyacrylamide gels. Larger fragment sizes were determined on 1.3 % agarose gels. Endonuclease Hind111 digests of A DNA and Hoe111 digests of 4 X 174 were used as size markers. To visualize the bands, gels were stained with ethidium bromide and photographed under ultraviolet light. The sizes of the fragments were determined from the photographic negatives using a Hewlett Packard 9872A plotter in digitizer mode coupled to an HP9845B computer. The computer program (Cozens, P. J., unpublished) constructs a standard curve from the size and mobilities of the standard fragments using the linearizing equation of Dr E. M. Southern (personal communication) and determines the size of the fragments in the experimental digests using this standard plot. Construction of Vitellogenin cDNA Clones
Vitellogenin cDNA clones were essentially constructed as described by Ohno et al. [lo] except that we used dG . dC tailing and the synthetic DNA was inserted into the PstI site of plasmid pBR322. All bacterial transformations were carried out as outlined by NIH guidelines; P3 physical and EK2 biological containment were employed. Positive Identification
of Vitellogenin cDNA Clones
Plasmid DNA binding to diazobenzyloxymethylcellulose paper, hybridization, elution of RNA from
DNA filters was done according to Alwine et al. [11] and Smith et al. [32]. The rabbit reticulocyte lysate cell-free system assay was done according to Pelham and Jackson [13]. Preparation of the antigen, immunization of rabbits and radioimmunoprecipitation were done according to Jost et al. [14,19]. Labelling qf m R N A
Vitellogenin mRNA was isolated as described by Jost et al. [14]. The partial hydrolysis of mRNA and labelling in vitro was performed as described by Maizels [15]. Fragments of about 200- 300 nucleotides with a specific activity of 5 x lo7 counts min-' pg-' were obtained. DNA-R N A Hyhridizat ion
Plasmid DNA covalently bound to diazobenzyloxymethyl-cellulose paper was hybridized with 32Plabelled RNA overnight at 42°C in 50"/, formamide, 5 x NaCl/Cit (NaCl/Cit is 0.15 M NaCI, 0.01 5 M trisodium citrate, pH 7.0). Filters were then washed for 2 h with several changes of 50 formamide, 5 x NaCl/Cit at 42°C and finally with 0.1 x NaCljCit at 65 "C for 1 h. The dried filters were exposed overnight to X-ray films.
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