SummaryXer site-specific recombination functions in the stable inheritance of circular plasmids and bacterial chromosomes. Two related recombinases, XerC and XerD, mediate this recombination, which 'undoes' the potential damage of homologous recombination. Xer recombination on natural plasmid sites is preferentially intramolecular, converting plasmid multimers to monomers. In contrast, recombination at the Escherichia coli recombination site, dif, occurs both intermolecularly and intramolecularly, at least when dif is inserted into a multicopy plasmid. Here the DNA sequence features of a family of core recombination sites in which the XerC-and XerD-binding sites, which are separated by 6 bp, were analysed in order to ascertain what determines whether recombination will be preferentially intramolecular, or will occur both within and between molecules. Sequence changes in either the XerC-or XerD-binding site can alter the recombination outcome. Preferential intramolecular recombination between a pair of recombination sites requires additional accessory DNA sequences and accessory recombination proteins and is correlated with reduced affinities of recombinase binding to recombination core sites, reduced XerC-mediated cleavage in vitro, and an apparent increased overall bending in recombinase-core-site complexes.

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Comparative screening of a library of cloned Drosophila DNA with polyadenylated mRNA from Drosophila adult head and adult body identified 20 cloned sequences expressed more abundantly in the tissues of the head than in other tissues. Quantitati.on using a "dot blot" hybridization assay demonstrated that the DNA sequences are expressed on adult head polysomes from 3 to 177 times more abundantly than on body polysomes, and their transcripts represent from 0.05 to 0.6656 of the polyadenylated mRNA mass of the head. The steady-state nuclear RNA concentrations of four species were (determined to be from 13 to 48 times greater in head nuclei than in body nuclei, an indication that their expression is controlled at least in part at the transcriptional level. The chromosomal locations of all 20 headspecific clones were identified by in situ mapping, and no distinct clustering was observed. However, four of the clones were shown by Southern and Northern blot analysis to contain multiple RNA coding sequences. The genes in these tightly clustered ssets were observed to be expressed simultaneously in some cases and differently in others.

Second thoracic mammary glands of immature BALB/c female mice were stimulated to pregnancy-like lobuloalveolar (LA) development after 6 days of incubation in a corticosteroid-free step I culture medium containing insulin, prolactin, estradiol, progesterone, and growth hormone. A low basal level (0.0009%) of casein mRNA (mRN4.,) sequences was detectable in the LA glands by a specific cDNA probe. Subsequent incubation of the LA glands for 3 days in medium containing insulin and prolactin or insulin and cortisol failed to elicit mRNAesn above the basal level, indicating that neither prolactin nor cortisol alone can support casein gene expression.However, an increase in mRNA. levels was observed when the 3-day incubation with insulin and cortisol or insulin and prolactin was followed by 3 days of culture in presence of insulin, prolactin, and cortisol. When a 3-day incubation with insulin and prolactin was followed by 3 days in insulin and cortisol medium, mRNAes. levels in the gland remained similar to the basal level. However, a 20-fold increase in the mRNAesn levels ensued when the LA glands were sequentially incubated for 3 days in insulin and cortisol and then for another 3 days in insulin and prolactin medium. After a preincubation in insulin and cortisol medium, the LA glands retained residual cortisol during subsequent incubation in insulin and prolactin medium, and the mRNAes. levels in these glands were related to the level of residual cortisol present. When mRNAesn and the residual cortisol level reached a minimum, addition of fresh cortisol to the medium caused a 20-fold increase in the mRNAesn levels. This indicates that cortisol is a limiting factor in insulin and prolactin medium and its presence is absolutely required for casein gene expression.The requirement that prolactin and cortisol be present for lactogenesis was delineated over 2 decades ago (1, 2). Since then, numerous studies have shown that, during lactation, a marked increase of mammary cell RNA and protein, including casein, is dependent upon stimulation by prolactin and cortisol (3, 4). We have shown that cortisol is required for the maintenance of casein-synthesizing polysomes, poly(A)+RNA synthesis, and casein mRNA (mRNAsn) accumulation in the lactating mammary gland of the mouse (5-7). Moreover, a regulatory action of the glucocorticoid on transcription of the casein gene in the mammary gland in vivo has been demonstrated (7). However, complexities in the animal limit elucidation of the discrete role of polypeptide and steroid hormones which regulate specific expression of the casein gene in breast cells.Fragments of mammary tissue from pregnant mice were demonstrated to be capable of synthesizing casein in a culture medium containing the lactogenic hormones plus insulin (8). This provided an in vitro model for studying molecular responses of the mammary cells to prolactin and glucocorticoid action in a chemically defined medium containing insulin, which is needed for viability of the mammary parenchyma in vitro (9). Subsequen...

Entire second thoracic mammary glands of estrogen- and progesterone-treated immature virgin BALB/c mice were stimulated to pregnancylike lobuloalveolar morphogenesis after 6 days of incubation with insulin (5 micrograms/ml), aldosterone (1 micrograms/ml), growth hormone (5 micrograms/ml), cortisol (5 micrograms/ml), and prolactin (80 ng/ml, present as a contaminant in 5 micrograms/ml growth hormone). The alveolar growth in the glands, as judged by morphological studies, was accompanied by an increase in cell number as a function of incubation time in the hormonal medium. Hybridization of the total RNA from these glands to the casein mRNA specific complementary DNA probe (cDNAcsn) revealed that the level of casein mRNA rises from 0.00012 to 0.005% between 1 and 6 days of incubation. Estimates showed that the concentration of casein mRNA per cell rises 17-fold from 70 molecules on Day 1 to 1200 molecules on Day 6, whereas the number of epithelial cells increases only twofold during the same incubation time. When the growth hormone preparation was totally replaced by 80 ng of prolactin during the 6-day incubation, casein-mRNA levels were found to be 0.0083%. These results demonstrate that a pregnancy-like morphogenesis and concurrent expression of the casein gene in vitro can be achieved in a controlled hormone environment containing high cortisol and low prolactin concentrations. This one-step mammogenesis-lactogenesis culture model should be useful for studying the mechanisms of hormonal regulation of casein-gene expression observed in prepartum mammary gland in vivo.