No abstract

Integration of mouse endogenous retroviral (MERV) elements is responsible for an estimated 10% of spontaneous mutations that have been characterized in the laboratory mouse. We recently identified a MERV integration in the first intron of the Zinc fingers and homeoboxes 2 (Zhx2) gene in BALB/cJ mice, resulting in reduced Zhx2 expression. This integration is found in BALB/cJ but not in other BALB/c substrains, indicating that it occurred after these substrains separated in the late 1930s. We have characterized this MERV element and show here that it belongs to the ETnII-alpha class of elements. Our analysis reveals that the Zhx2 ETn element lacks a 69-bp sequence compared to most other ETn elements which may be due to recombination between two identical 13-bp elements. Three mature Zhx2 transcripts are found in the liver of BALB/cJ mice. The major transcript is spliced from Zhx2 exon 1 to the 5' ETn LTR and is polyadenylated at the 3' LTR. Of the two less abundant transcripts, one is identical to the wild-type transcript, whereas the second contains 183 bp of ETn sequence spliced between Zhx2 exons 1 and 2. We have also sequenced and analyzed products from the fas ( lpr ) ETn found in MRL/lpr mice and show that it belongs to the ETnII-beta class of elements.

␣-Fetoprotein (AFP) transcription is activated early in hepatogenesis, but is dramatically repressed within several weeks after birth. AFP regulation is governed by multiple elements including three enhancers termed EI, EII, and EIII. All three AFP enhancers continue to be active in the adult liver, where EI and EII exhibit high levels of activity in pericentral hepatocytes with a gradual reduction in activity in a pericentral-periportal direction. In contrast to these two enhancers, EIII activity is highly restricted to a layer of cells surrounding the central veins. To test models that could account for position-dependent EIII activity in the adult liver, we have analyzed transgenes in which AFP enhancers EII and EIII were linked together. Our results indicate that the activity of EIII is dominant over that of EII, indicating that EIII is a potent negative regulatory element in all hepatocytes except those encircling the central veins. We have localized this negative activity to a 340-bp fragment. This suggests that enhancer III may be involved in postnatal AFP repression.T he ␣-fetoprotein (AFP) gene, which encodes the major serum protein in the developing mammalian fetus, is transcribed in the yolk sac visceral endoderm, fetal liver, and, to a much lesser extent, in the fetal gut and kidney (1). AFP activation during hepatogenesis occurs as primordial hepatocytes migrate out from the developing foregut (2). The AFP gene continues to be expressed abundantly in hepatocytes during development, but is dramatically repressed postnatally (3); in the liver, this represents a nearly 10,000-fold reduction in transcription (3). The AFP gene is normally expressed at extremely low levels in the adult liver, but can be reactivated during periods of renewed cell growth such as during liver regeneration and in hepatocellular carcinomas (4).Perinatal AFP repression does not occur uniformly in all hepatocytes. Rather, reduced AFP mRNA levels are first seen in hepatocytes that reside in the perivenous regions of the liver, i.e., those cells surrounding the portal triads. AFP repression continues in a gradient-like fashion in a perivenous to pericentral direction (5, 6). Thus, the last cells to express AFP before complete shut-off reside in a single layer of hepatocytes surrounding the central veins. In addition to AFP repression, other transcriptional changes occur in the perinatal liver. In particular, the mRNAs for numerous liver enzymes become zonally expressed, i.e., synthesized exclusively in pericentral or perivenous hepatocytes (reviewed in ref. 7). For example, glutamine synthetase and ornithine aminotransferase are expressed exclusively in a narrow band of cells surrounding the central veins (8-10).Other enzymes, such as carbamoylphosphate synthetase and ornithine transcarbamylase, are expressed in a broad band of hepatocytes encircling the portal triads (10, 11). These periportal and pericentral regions of expression are nonoverlapping. The basis for this position-dependent regulation is not known, but a mathematica...

Estrogen plays an important role in skeletal physiology by maintaining a remodeling balance between the activity of osteoblasts and osteoclasts. In an attempt to decipher the mechanism through which estrogen elicits its action on osteoblasts, experimentation necessitated the development of a culturing environment reduced in estrogenic compounds. The selected medium (OPTI-MEM) is enriched to sustain cultures under reduced fetal bovine serum (FBS) conditions and is devoid of the pH indicator phenol red, a suspected estrogenic agent. This protocol reduced the concentration of FBS supplementation to 0% through successive 24 h incubations with diminishing amounts of total FBS (1%, 0.1%, and 0%). The protocol does not appear to alter the viability, cell morphology, or osteoblast-like phenotype of 7F2 and UMR-106 cell lines when compared with control cells grown in various concentrations of FBS. Although the rate of mitotic divisions declined, the 7F2 and UMR-106 cultures continued to express osteoblast-specific markers and exhibited estrogen responsiveness. These experimental findings demonstrate that the culture protocol developed did not alter the osteoblast nature of the cell lines and provides a model system to study estrogen's antiresorptive role on skeletal turnover.

No abstract