Mammary gland-specific hypomethylation of Hpa II sites flanking the bovine alpha S1-casein gene.

In the lactating cow, mammary gland-specific hypomethylation occurs at two Hpa II sites in the 5'-flanking region of the alpha S1-casein gene, and one in the 3'-region. These sites, A, B and C, are at nucleotide position -1388, -774 and +18034, respectively, relative to the major transcription start site. Site B was hypomethylated when the alpha S1-casein gene was expressed, and methylated when not expressed. In transgenic mice containing the bovine alpha S1-casein 5' and 3' regulatory elements fused to the human lactoferrin (hLF) cDNA, in some cases similar methylation patterns of sites A and B, as compared to the situation in the cow, were observed. In five mouse lines (out of the seven analysed) expressing the transgene in the milk, site B was hypomethylated in the mammary gland, while it was methylated in liver. In the two other mouse lines, no correlation was found between transgene expression and mammary gland-specific hypomethylation of site B. One of the five mouse lines with transgene expression and showing mammary-gland-specific hypomethylation of site B was studied in detail. In this mouse line, induction of transgene expression preceded hypomethylation of site B.

Expression of human lactoferrin in milk of transgenic mice.

The expression of human lactoferrin (hLF) in the milk of transgenic mice is described. Regulatory sequences derived from the bovine alpha S1-casein gene were fused to the coding sequence of the hLF cDNA and several lines of transgenic mice were generated. Human LF RNA was detected exclusively in the mammary gland of lactating females and only after the onset of lactation. No aberrant RNA products could be detected using northern blotting and primer extension analysis. The hLF concentrations in the milk ranged from less than 0.1 to 36 micrograms ml-1. Human LF thus expressed did not differ from human milk derived LF, with respect to molecular mass and immunoreactivity with monoclonal and polyclonal antibodies.

Efficient generation of functional transgenes by homologous recombination in murine zygotes.

To assess the feasibility of generating functional transgenes directly via homologous recombination between microinjected DNA fragments, three overlapping genomic DNA fragments, together constituting the human serum albumin (hSA) gene, were coinjected into murine zygotes. The resulting transgenic mice were analyzed for structure and expression of the transgene. All transgenic mice carried recombined hSA DNA fragments and 74% contained a reconstituted hSA gene. HSA expression could be detected in liver and serum in most (72%) of these animals. Only correctly sized hSA transcripts were observed. Transgenic hSA could not be distinguished from the human serum-derived protein by radioimmunoassay or Western blotting. The high frequency and accuracy of homologous recombination in murine zygotes reported here allows the efficient generation of relatively large transgenes.

Generation of transgenic dairy cattle using 'in vitro' embryo production.

We have combined gene transfer, by microinjection, with 'in vitro' embryo production technology, enabling us to carry out non-surgical transfer, to recipient cows, of microinjected embryos that have been cultured from immature oocytes. Using this approach, we have established 21 pregnancies from which 19 calves were born. Southern blot analysis proved that in two cases the microinjected DNA had been integrated in the host genome.

Adenovirus VAI-RNA regulates gene expression by controlling stability of ribosome-bound RNAs.

Adenovirus VAI-RNA is a small virally encoded RNA that is required for efficient protein synthesis at late times of adenoviral infection. We show that in transient transfection assays VAI-RNA promotes not only an increased level of protein encoded by a co-transfected marker (CAT) plasmid, but also a marked accumulation of its transcript. The increases in CAT protein and RNA levels reflect an enhanced stability of the cytoplasmic RNA as shown by primer extension analyses of RNA isolated from transfected cells upon transcriptional arrest. Surprisingly, the ability of VAI to activate expression of CAT requires the translation of a substantial portion of the RNA: when translation is prevented by elimination of the initiator AUG codon or by introduction of stop codons 5' to codon 107, VAI-RNA is no longer capable of increasing CAT RNA levels; the introduction of stop codons 3' of codon-135, on the other hand, does not significantly impair VAI-RNA function. We conclude that in addition to its role as a specific activator of translation, adenovirus VA genes function to regulate the stability of ribosome-bound RNA.

Conserved sequence motifs upstream from the co-ordinately expressed vitellogenin and apoVLDLII genes of chicken.

The vitellogenin and apoVLDLII yolk protein genes of chicken are transcribed in the liver upon estrogenization. To get information on putative regulatory elements, we compared more than 2 kb of their 5' flanking DNA sequences. Common sequence motifs were found in regions exhibiting estrogen-induced changes in chromatin structure. Stretches of alternating pyrimidines and purines of about 30-nucleotides long are present at roughly similar positions. A distinct box of sequence homology in the chicken genes also appears to be present at a similar position in front of the vitellogenin genes of Xenopus laevis, but is absent from the estrogen-responsive egg-white protein genes expressed in the oviduct. In front of the vitellogenin (position -595) and the VLDLII gene (position -548), a DNA element of about 300 base-pairs was found, which possesses structural characteristics of a mobile genetic element and bears homology to the transposon-like Vi element of Xenopus laevis.

Estradiol-dependent transcription initiation upstream from the chicken apoVLDLII gene coding for the very-low-density apolipoprotein II.

We have investigated RNAs originating from the 5'-flanking region of the chicken very-low-density apolipoprotein II (apoVLDLII) gene. S1 nuclease mapping and primer extension experiments revealed two minor upstream transcription start points located 1105 and 1530 nucleotides in front of the apoVLDLII gene. Transcription starting at these points is dependent upon estradiol as is transcription from the major start points. The transcripts are polyadenylated, but are not detectable in polysomes. Run-on assays indicated that the low concentration of the upstream initiated transcripts is due both to low transcription levels and to low transcript stability. The sequence around the upstream start points does not show strong homologies with consensus sequences of promoters for eukaryotic protein encoding genes. Nevertheless, the upstream sequences are transcribed in vivo by RNA polymerase II.

The nucleotide sequence of the chicken apo very low density lipoprotein II gene.

The nucleotide sequence of the chicken apo Very Low Density Lipoprotein II (apoVLDL II) gene and the regions immediately flanking the gene was determined. Nuclease S1 mapping showed that transcription is initiated at two sites, about 11 bp apart, of which the one lying downstream is used preferentially. Comparison of the 2918-base pair gene sequence with the earlier determined cDNA sequence [Wieringa et al. (1981) Nucleic Acids Research 9, 489-501] enabled us to identify the four exons which are 38 (or 49), 100, 160 and 358 bp long. One of the intron-exon junctions has an unusual sequence. In the 5' flanking region several palindromic sequences are observed. Sequences near the 5' and 3' ends show homologies with the ovalbumin gene.