Cell-specificity of the chicken ovalbumin and conalbumin promoters.

A series of recombinant plasmids containing increasing lengths of the 5'-flanking promoter sequences of the chicken conalbumin and ovalbumin genes fused to the sequences coding for the SV40 T-antigen have been constructed. These recombinants were introduced into a variety of established cell lines and primary cultured cells by nuclear microinjection. Promoter activity was estimated by monitoring T-antigen synthesis by indirect immunofluorescence. We show that the microinjected ovalbumin and conalbumin promoter regions do not function in chicken fibroblasts, kidney cells and in a variety of non-chicken cells, irrespective of the presence of steroid hormone receptors. In contrast, these promoter regions are active in primary cultured chicken embryonic hepatocytes and oviduct tubular gland cells, suggesting the presence of cell-specific transcription factors in these cells. Unexpectedly, promoter sequences close to the TATA boxes of both the ovalbumin and conalbumin genes are sufficient to confer cell-specific expression. Most of the controls exerted on the ovalbumin and conalbumin promoters in the whole animal appear to be reproduced in vitro by nuclear microinjection of the chimeric genes into the primary cultured cells. However, the microinjected ovalbumin promoter is active in embryonic hepatocytes and thus escapes the regulation imposed on the corresponding inactive endogenous gene.

Chicken oviduct progesterone receptor: location of specific regions of high-affinity binding in cloned DNA fragments of hormone-responsive genes.

We have used a DNA-cellulose competitive binding assay to measure the extent of displacement of the chicken oviduct progesterone-receptor complex from calf thymus DNA-cellulose by purified cloned fragments of genomic DNA. Several DNA fragments from hormonally responsive genes coding for egg-white proteins were found to be efficient competitors for either crude or partially purified receptor complexes when compared with calf thymus DNA. Data obtained with deletion mutations constructed in vitro allowed delineation of a specific region necessary for strong competition, located 250--300 bp upstream from the mRNA startsite of the ovalbumin gene. Sequence homologies with this 5'-upstream region were observed in other fragments of the ovalbumin, conalbumin, ovomucoid, X and Y genes, which were also efficient competitors. Based on a comparison of such sequences of homology, a consensus sequence that may constitute a region binding progesterone-receptor complex has been constructed: ATCCCTTATTATTCTGGTTTGTA. The results suggest that specific double-stranded DNA sequences are recognized by the oviduct progesterone-receptor complex in vitro, and are relevant to the question of whether specific DNA sequences are directly involved as genomic binding sites for steroid receptors.

The chicken conalbumin gene: studies of the organization of cloned DNAs.

The construction of a double-stranded conalbumin cDNA plasmid (1) has allowed us to investigate the structure of the conalbumin gene. Restriction enzyme mapping of chicken genomic DNA reveals that the conalbumin gene is split and is contained in three EcoRI fragments "a", "b", "b" and "c" which have sizes of 10.7, 4 and 2.5 kb, respectively. Analysis with specific probes shows that the orientation of these fragments with respect to transcription is 5'-"b", "c" and "a"-3. The fragments Eco "b", Eco "c" and part of Eco "a" have been isolated by molecular cloning from three different "libraries". Electron microscopic studies of hybrids between cloned DNA's and conalbumin mRNA show that one of the isolated clones, lambda C4-conl, contains the coding sequences for the first 940 nucleotides of the mRNA (out of 2400). This region is highly split, since it contains seven short exonic sequences separated by six intervening sequences. The DNA region coding for these 940 nucleotides is 5 times longer than the mRNA coding sequences, a ratio very similar to that found for other chicken genes.

The ovalbumin gene region: common features in the organisation of three genes expressed in chicken oviduct under hormonal control.

Two large DNA fragments overlapping the chicken ovalbumin gene have been isolated by molecular cloning. Analysis of these fragments provided a map of a 46,000-base pair region of the chicken genome. This region contains the complete ovalbumin gene (including its mRNA leader-coding sequence) and at least two other genes of unknown function. All three genes are orientated in the same direction and their expression in chicken oviduct is under hormonal control. The three genes share some sequence homologies, suggesting that duplications have occurred in the ovalbumin gene region in the course of evolution.

Organisation and sequences at the 5' end of a cloned complete ovalbumin gene.

A clone which contains the complete chicken ovalbumin gene, including its leader coding sequences, has been isolated. From electron microscopic analysis of this DNA we conclude that the minimal size of the transcriptional unit for ovalbumin is 7.7 kilobases. The DNA sequence of the region surrounding the 5' end of the ovalbumin gene is presented. Comparison of this sequence with those of other eukaryotic genes reveals striking similarities, possibly related to a promoter region, approximately 30 base pairs upstream from the site coding for the 5' end of the mRNA.

The ovalbumin split gene: molecular cloning of Eco RI fragments "c" and "d".

The Eco RI fragments "c" and "d" of the ovalbumin gene (1, 2) have been isolated by molecular cloning. Restriction enzyme mapping and electron microscopy have confirmed that the two fragments contain the same ovalbumin mRNA coding sequences. These sequences are split into two regions which have been mapped in fragments "c" and "d". There is no evidence that the ovalbumin mRNA sequences contained in these fragments could be further interrupted. Our results confirm that the presence of Eco RI fragment "d" in some chickens is due to the existence of an allelic variant of the ovalbumin gene which contains an additional Eco RI site within the region corresponding to Eco RI fragment "c". This additional Eco RI site appears to be the main difference between the two alleles. Finally, our results provide a direct demonstration that most of the ovalbumin mRNA sequences are encoded for by Eco RI fragments "a", "b" and "c".

Isolation by molecular cloning of a fragment in the split ovalbumin gene.

An EcoRI fragment of chicken DNA (fragment 'a') containing a sequence complementary to the 3' half of ovalbumin mRNA has been isolated by molecular cloning. Analysis of the cloned fragment proves conclusively that the chicken ovalbumin gene is split. Fragment 'a' contains no extensive sequence repeated elsewhere in the genome and represents the only type of organisation of this part of the split ovalbumin gene in chicken genome.