RESUMO
Purified mRNA coding for chicken vitellogenin, a precursor of egg yolk proteins, was transcribed to complementary DNA (cDNAvit) with avian myeloblastosis virus (AMV) reverse transcriptase. Double-stranded cDNA was synthesized with Escherichia coli DNA polymerase I (fragment A) using the self priming ability of the cDNA. Following S1 nuclease digestion the double-stranded cDNA was inserted into the Hind III site of plasmid pBR322 using the poly(dA) . poly(dT) tailing method, and the hybrid molecules were used to transform Escherichia coli chi 1776. Ampicillin-resistant colonies were screened by colony hybridization with 125I-labeled vitellogenen mRNA. Further screening of positive clones was done by agarose gel electrophoresis and in situ hybridization with 125I-labeled vitellogenin mRNA. In addition, plasmid DNA covalently bound to diazotized paper was used to select complementary mRNA sequences. The cloned vitellogenin sequences were shown to hybridize to a mRNA which directs the synthesis of immunoprecipitable vitellogenin when translated in a reticulocyte lysate cell-free system. The length of the inserted cDNA was determined by agarose gel electrophoresis and heteroduplex mapping. The largest insertion was about 2500 base pairs. Restriction mapping indicates that at least three plasmids out of four have different sequences.
Assuntos
DNA/biossíntese , Lipoproteínas/genética , Vitelogeninas/genética , Animais , Galinhas , Mapeamento Cromossômico , Clonagem Molecular , DNA/análise , Elementos de DNA Transponíveis , DNA Recombinante/biossíntese , Escherichia coli/genética , Genes , Plasmídeos , RNA Mensageiro/genética , Transcrição Gênica , Transformação BacterianaRESUMO
Two phage lambda recombinant DNA clones covering the entire sequence of an avian vitellogenin gene, plus flanking regions, have been isolated from an erythrocyte DNA gene library and characterized by R-loop and restriction mapping. The total length of this avian vitellogenin gene is 23 kb. The cloned sequences flanking the gene at the 5' and 3' end are 7 and 3 kb, respectively. The total length of exons in the two clones is 6.7 kb (vitellogenin mRNA is 6.6 kb). The gene is interrupted by at least 25 introns with a mean intron length of 940 bp. Some 6--10 additional very small introns may also be present but they were not observed reproducibly. The mean exon length is 250 bp. Restriction endonuclease digests of total liver genomic DNA and lambda recombinant DNA were also analyzed by electrophoresis. Southern blotting and hybridization with cloned vitellogenin cDNA. The results show an identity of organisation of this vitellogenin in the DNA from the two sources, thus ruling out a possible cloning artifact. In contrast to Xenopus vitellogenin we have found no evidence to suggest that avian vitellogenin is encoded by a small family of related genes.
Assuntos
Galinhas/genética , Genes , Lipoproteínas/genética , RNA Mensageiro/genética , Vitelogeninas/genética , Animais , Bacteriófago lambda/genética , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Estrogênios/farmacologia , Microscopia Eletrônica , Hibridização de Ácido Nucleico , Transcrição Gênica/efeitos dos fármacosRESUMO
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)]. 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 laevis, 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 appear to 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.
Assuntos
Clonagem Molecular , DNA/genética , Lipoproteínas/genética , RNA Mensageiro/genética , Vitelogeninas/genética , Animais , Sequência de Bases , Galinhas , DNA/metabolismo , Enzimas de Restrição do DNA , Hibridização de Ácido Nucleico , Plasmídeos , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Vitelogeninas/biossíntese , Xenopus laevisRESUMO
The two isoschizomeric restriction endonucleases Msp I and Hpa II have been used to study changes in the methylation pattern of the vitellogenin gene after estrogen treatment. In the liver of the both estrogen-treated and nontreated chickens the vitellogenin gene is heavily methylated. However, estradiol causes a demethylation of a Hpa II site(s) at the 5' end region of the gene. The same Hpa II restriction site(s) is also unmethylated in the liver of egg laying hens but is methylated in the liver of roosters or in erythrocyte DNA. In the mature oviduct where no vitellogenin is synthesized but where several egg white proteins are under the control of estrogen, there is also a demethylation of the 5'end region of the vitellogenin gene. This demethylation does not precede the initiation of transcription of the gene but persists, and increases, even after vitellogenin transcription has ceased in the liver. Taken together, the results strongly suggest that in estrogen-responsive tissues the vitellogenin gene becomes demethylated at its 5' end region after estrogen stimulation whether the gene is expressed or not.