Isolation and fine structure organisation of an avian vitellogenin gene coding for the major estrogen-inducible mRNA

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.     

Evidence for a role of topoisomerase II in the Ca2+-dependent basal level expression of the rat prolactin gene

The PRL gene is expressed at a high basal level in rat pituitary tumor GH3 cells, and this basal level enhancement of PRL gene expression is maintained through a Ca2+-calmodulin-dependent mechanism. We have now examined whether the enzyme, DNA topoisomerase II, which has been shown to be phosphorylated by a Ca2+-calmodulin-dependent protein kinase, plays a role in the Ca2+-calmodulin-dependent basal level enhancement of PRL gene expression. The topoisomerase II inhibitor, novobiocin, at concentrations in the range of 35-140 microM, effectively blocked the ability of Ca2+ to increase PRL mRNA levels. Examination of the effects of novobiocin on the levels of protein synthesis, glucose-regulated protein (GRP) 78 mRNA, histone 3 mRNA, and 18S ribosomal RNA indicated that the drug selectivity inhibited PRL gene expression. Two other topoisomerase II inhibitors, m-AMSA and VM26, also diminished the Ca2+-induced levels of PRL mRNA at concentrations (100-400 nM) that did not lower total mRNA levels. We then examined whether topoisomerase II interacted nonrandomly with DNA from the 5' transcribed and 5'-flanking region of the rat PRL gene by in vitro mapping of topoisomerase II DNA cleavage sites. In initial assays with a 10.5 kilobase (kb) PRL genomic DNA fragment containing 3.5 kb of 5'-transcribed DNA and 7 kb of 5'-flanking DNA, we detected 4 major cleavage sites in the following regions: site 1, +1500 to +1600; site 2, +1 to -100; site 3, -1200 to -1300; and site 4, -2900 to -3000.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the nuclear encoded OEE1 protein is required for oxygen evolution and stability of photosystem II particles in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii the oxygen evolving enhancer protein 1 (OEE1), which is part of the oxygen evolving complex of photosystem II (PS II), is coded for by a single nuclear gene (psb1). The nuclear mutant FuD44 specifically lacks the OEE1 polypeptide and is completely deficient in photosynthetic oxygen evolution. In this mutant a 5 kb DNA insertion into the 5' region of the psb1 gene results in the complete absence of OEE1 mRNA and protein. A revertant, FuD44-R 2, which is capable of 30% of the photosynthetic oxygen evolution of wild-type cells, has lost 4 kb of the 5 kb DNA insert, and accumulates both OEE1 mRNA and protein, although at levels somewhat less than those of wild-type cells. Absence of the OEE1 protein in the FuD44 mutant does not affect the accumulation of other nuclear encoded PS II peripheral polypeptides. OEE1 absence does, however, result in a more rapid turnover of the chloroplast encoded PS II core polypeptides, thus resulting in a substantial deficiency of PS II core polypeptides in FuD44 cells. These PS II core proteins again accumulate in revertant FuD44-R2 cells.     

A single gene encoding vitellogenin in the sea urchin Strongylocentrotus purpuratus: sequence at the 5'' end.

The synthesis of vitellogenin (yolk protein precursor) in the sea urchin, Strongylocentrotus purpuratus, is unique in that both males and females produce a high level of the protein. In this paper we show that this organism also is unique in possessing only a single vitellogenin gene. Like the genes that encode analogous proteins in vertebrates, the sea urchin gene is large, about 19 kb in length. The sequence surrounding the 5' end of the gene revealed several other similarities to vertebrate vitellogenin genes: the signal sequence is exceptionally short and has a sequence similar to those from frog and chick; there is a canonical TATA box at -32; and there is a sequence closely resembling the estrogen-responsive element at -207.     

Isolation and characterization of genomic clones covering the chicken vitellogenin gene

A series of overlapping recombinant clones, which cover the vitellogenin gene, has been isolated from a phage-lambda linked chicken gene library. The DNA of the overlapping clones spans 28 kb of contiguous DNA sequences in the chicken genome. Electron microscopic analysis of hybrids between vitellogenin mRNA and the genomic clones indicates that the chicken vitellogenin gene has a length of approximately 22 kb, about 3.8 times the size of the mRNA. The mRNA sequence is interrupted by at least 33 intervening sequences (introns). Comparison with the vitellogenin gene A2 from Xenopus laevis (Wahli et al., 1980, Cell 20: 107-117) indicates conservation of the number and length of the exons during evolution. Heteroduplex analysis reveals a short stretch of sequence homology between the genes from chicken and frog.     

Human 2-5A synthetase: characterization of a novel cDNA and corresponding gene structure.

The enzyme 2-5A synthetase is induced in cultured cells in response to interferon (IFN) treatment. A lambda gt10 cDNA library of mRNA from IFN-induced Daudi lymphoblastoid cells was screened with oligonucleotide probes. Several overlapping cDNAs were isolated and shown to be derived from the human synthetase gene using filter selection and oocyte microinjection assays. The nucleotide sequence of one of these, cDNA 8-2, extended the 2-5A synthetase sequence already described 72 bp in the 5' direction but was found to differ significantly in coding sequence at the 3' end. The longest cDNA isolated (6-2) was approximately 1.4 kb. By Northern hybridization analysis single mRNAs of 1.7 kb were detected in Daudi and T98G (glioblastoma) cells. However, in HeLa cells, four mRNAs ranging in size from 1.5 to 3.5 kb were found, one of which differed at the 3' end. Analysis of both phage and cosmid genomic clones and comparison with genomic DNA indicate that there is a single gene for 2-5A synthetase, comprising at least six exons and five introns, which can undergo a novel form of alternative RNA processing depending on cell type.     

Sequence homologies within the 5'' end region of the estrogen-controlled vitellogenin gene in Xenopus and chicken.

In oviparous vertebrates vitellogenin, the precursor of the major yolk proteins, is synthesized in the liver of mature females under the control of estrogen. We have established the organization and primary structure of the 5' end region of the Xenopus laevis vitellogenin A2 gene and of the major chicken vitellogenin gene. The first three homologous exons have exactly the same length in both species, namely 53, 21 and 152 nucleotides, and present an overall sequence homology of 60%. In both species, the 5'-non-coding region of the vitellogenin mRNA measures only 13 nucleotides, nine of which are conserved. In contrast, the corresponding introns of the Xenopus and the chicken vitellogenin gene show no significant sequence homology. Within the 500 nucleotides preceding the 5' end of the genes, at least six blocks with sequence homologies of greater than 70% were detected. It remains to be demonstrated which of these conserved sequences, if any, are involved in the hormone-regulated expression of the vitellogenin genes.     

The human thyroglobulin gene is over 300 kb long and contains introns of up to 64 kb.

Thyroglobulin (Tg), the precursor of thyroid hormones, is a 660.000 Da dimeric glycoprotein synthesized exclusively in the thyroid gland. We have cloned the human thyroglobulin gene from cosmid and phage libraries and constructed a complete restriction map. The gene encodes an 8.7 kb mRNA, covers at least 300 kb DNA and contains at least 37 exons separated by large introns of up to 64 kb. A striking difference in structure between the 5' and 3' part of the gene suggests that it is composed of two evolutionarily different regions. The first 30 kb DNA encode 3 kb of the mRNA, yielding an exon:intron ratio of 1:10, whereas the remaining 270 kb encodes 5.7 kb of the mRNA with an exon:intron ratio of 1:47. In thyroid cells, the Tg gene is not rearranged and nuclear RNA homologous with sequences internal to the 64 kb intron is present, suggesting that the Tg gene is transcribed as a 300 kb RNA.     

Age-associated changes in DNA methylation and mRNA level of the c-myc gene in spleen and liver of mice

Age-associated changes in DNA structure and mRNA level were studied for the c-myc gene in spleen and liver of mice at 2, 14 and 26 months of age. Neither amplification nor rearrangement of the gene was detected in either tissue at any age. However, a significant alteration was observed in the methylation profile. The profile of the gene and its vicinity was determined using various methylation-sensitive restriction enzymes. In both tissues, the gene had an unmethylated domain ranging from -2 kb upstream of the 5' end of the first exon to the 3' end of the first intron. It was flanked by partially methylated regions, where age-dependent changes as well as tissue specificity were observed. In the spleen, age-related hypomethylation was observed at both the 3' and 5' sides of the domain. In contrast, hypermethylation was found in the liver only at the 3' side. The steady-state level of c-myc mRNA showed a drastic decrease in liver from youth to middle age, while splenic mRNA changed little. The correlation between the changes of mRNA and DNA methylation is discussed.