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The multigene family which codes for the mouse major urinary proteins consists of about 35 genes. Most of these are members of two distinct groups, group 1 and group 2. The group 1 and group 2 genes are organized in head-to-head pairs within 12 to 15 remarkably uniform chromosomal units or domains about 45 kilobase pairs (kb) in size. The 45-kb units are located on chromosome 4, and many of them are adjacent to each other. We propose that the 45-kb unit is a unit both of organization and of evolutionary change. In this study the homologies within the unit were observed by examining, in an electron microscope, heteroduplex and foldback structures made from cloned major urinary protein genes. These show that the 45-kb unit is a gigantic imperfect palindrome. Each arm of the palindrome contains two regions of inverted symmetry of 9.5 and 4.5 kb separated by a 3-kb nonsymmetrical region. We argue that the nonsymmetrical regions arose by a series of deletion events in the two arms of the palindrome. The center of the 45-kb unit is an 8-kb sequence without inverted symmetry flanked by the 9.5-kb regions, which contain the 4-kb genes and their immediate 5' and 3' flanking regions. The junction between adjacent 45-kb units is a 2- to 4-kb sequence without inverted symmetry flanked by the 4.5-kb regions. Some of the 45-kb units are arranged as direct tandem repeats. Others appear to be in inverted orientation with respect to a neighboring unit. Cloned major urinary protein genes show few incidences of the repetitive elements B1, B2, R, and MIF. Two elements, a B1 and an R, may be a constant feature of the 45-kb units. If so, in those cases in which the units are in tandem array, both of these elements will occur with a 45-kb periodicity. A comparison of corresponding parts of different 45-kb units shows that they differ because of a number of deletion or insertion events, particularly in the regions 3' to the genes.

Culture conditions which maintain hepatocytes in their in vivo state are not known. This hampers the study of liver gene expression and of direct responses of liver genes to hormonal stimulation. We argued that hepatocytes that were unable to divide might retain in vivo characteristics. We therefore plated mouse (BALB/c) hepatocytes on plastic dishes in medium lacking arginine and measured the levels and transcription rates of six tissue-specific mRNAs over a period of days. Alpha-fetoprotein mRNA began to accumulate at about 48 h of culture, and transcription could sometimes be detected after 72 h. The levels and transcription rates of four mRNAs (albumin, alpha-1-antitrypsin, apolipoprotein A1, and major urinary protein [MUP]) fell sharply. The rate of transcription of transferrin mRNA fell less rapidly, and its level remained high, partly due to its longer half-life. The overall pattern of gene expression in the plated cells did not exactly parallel that of either fetal or regenerating liver. The hepatocytes remained responsive to hormonal stimulation. Insulin and dexamethasone each tended to counteract changes in mRNA levels, for example, preventing the accumulation of alpha-fetoprotein mRNA. The effects of insulin were primarily due to changes in transcription rates. Bovine growth hormone and thyroxine elevated the levels of most of the mRNAs. Many of the effects of these hormones, when added singly, could not be ascribed to changes in transcription. The level of MUP mRNA was strongly affected by added hormones. The mRNA level at 5 days was increased by added insulin, dexamethasone, growth hormone, and thyroxine. In the presence of these three hormones, the decay in the transcription rate of the MUP genes was reduced about 10-fold. We conclude that hepatocytes plated under these nongrowing conditions can provide insights into the hormonal responsiveness of tissue-specific genes.