Temporal expression of retinoic acid receptors in hamster fetus during organogenesis and alteration by retinoic acid treatment.

Retinoic acid receptors alpha, beta and gamma (RAR alpha, beta, gamma) mRNAs from whole 8- to 15-day-old hamster fetuses were characterized and quantitated by Northern blots and solution hybridization using riboprobes from cloned hamster RAR cDNAs, derived from 12-day fetal hamster library. Two RAR alpha transcripts of approximately 3.1 and approximately 3.5 kb, one transcript of RAR beta approximately 2.8 kb and one transcript of RAR gamma approximately 3.1 kb were observed. The relative abundance levels of these transcripts were RAR gamma > beta > alpha. RAR beta and gamma levels peaked at day 11, increasing approximately 4-fold (beta) and approximately 2.5-fold (gamma) above their initial values at day 8. RAR alpha did not change appreciably and peaked on day 14 at 1.7 x of its lowest level at day 9. Regulation patterns of the three RARs diverged between days 8 and 9 and 13 and 14 postcoitum (p.c.) and coordinately increased between days 9 and 13 and decreased between days 14 and 15 p.c. In 12-day-old conceptuses exposed to all-trans-retinoic acid, RAR alpha did not increase significantly, but RAR beta increased 12-fold at 4 hr and RAR gamma 2-fold at 1 hr after the maternal treatments.

Genomic sequences capable of committing mouse and rat fibroblasts to adipogenesis.

The mouse Swiss 3T3-F442A/3T3-C2 cell system is well suited for the isolation of genes involved in commitment to adipogenesis. 3T3-F442A cells convert to adipocytes with high efficiency in response to confluence and insulin. The sister clonal line 3T3-C2 does not respond to these signals, but can convert to adipocytes when transfected with DNA from 3T3-F442A preadipocytes or from human fat. Human fat-tissue biopsy FO46 DNA transfected into 3T3-C2 gave rise to fat foci after two rounds of transfection and selection. A cosmid library of a subclone of secondary transfectant 3T3-C2/FO46-1 was screened for the human repetitive Alu sequence. Five out of eight Alu+ recombinant clones committed 3T3-C2 cells to adipogenesis. The adipose commitment (AC) activity of one cosmid, p18A4, was found to reside in two small, non-identical, subcloned sequences 1.2kb and 2.0kb in length, each separately able to commit 3T3-C2, precrisis mouse and rat fibroblasts and the multipotential C3H10T1/2 cell line to adipogenesis. We conclude that commitment to adipogenesis can be effected in vitro with high efficiency by transfection of specific sequences into a variety of host cells.

Alpha interferon suppresses hepatitis B virus enhancer activity and reduces viral gene transcription.

Interferons inhibit replication of hepatitis B virus (HBV). The mechanism for this inhibition was investigated by analyzing the effect of interferons on transcription of a chloramphenicol acetyltransferase reporter gene under control of HBV regulatory sequences and by determining the steady-state level of viral mRNAs in permanently HBV-transfected HepG2 cells. Low doses (100 U/ml) of alpha interferon (IFN-alpha) but not IFN-gamma inhibited chloramphenicol acetyltransferase expression in cultured cells transfected with plasmids containing the HBV enhancer linked to either HBV or simian virus 40 promoters. IFN-alpha also lowered expression of HBV mRNA in HBV-transfected HepG2 cells actively replicating virus, suggesting that IFN-alpha inhibits HBV replication by reducing transcription of viral genes driven by the HBV enhancer.

Commitment of mouse fibroblasts to adipocyte differentiation by DNA transfection.

Cells of the mouse cell line 3T3-F442A can be induced by various hormones to differentiate into adipocytes, whereas cells of 3T3-C2, a subclone of 3T3, cannot. However, transfection of DNA from uninduced 3T3-F422A cells into 3T3-C2 cells permits recovery of 3T3-C2 transfectants that differentiate into adipocytes in the presence of insulin. DNA isolated from human fat tissue, when transfected into 3T3-C2 mouse cells, also gives rise to mouse transfectants that are induced to differentiate into adipocytes by the addition of insulin. Apparently, transfection of a trans-regulatory gene (or genes) from 3T3-F442A or human fat cells into 3T3-C2 cells is sufficient to commit 3T3-C2 cells to adipocyte differentiation.

Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes.

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.

Growth-dependent expression of a cell surface glycoprotein.

The expression of the hepatocellular membrane receptor for desialylated galactose-termining glycoproteins was studied during different proliferative stages of a human hepatoma cell line. Rapidly growing cells exhibited a reduced endocytotic rate of desialylated orsomucoid as compared to non-growing cells. This reduction was shown to be the consequence of a lower concentration of active cell-surface associated receptor protein in the dividing cells.

Complementation of gene deletions by cell hybridization.

Overlapping deletions in chromosome 7 of the mouse are responsible for activity deficiencies of various liver-specific enzymes, including tyrosine aminotransferase (TAT). In an effort to elucidate the nature and type of action of the deleted genes, somatic cell hybridization experiments were carried out. Enzyme-deficient liver cells of homozygous mutant mice or normal liver cells of control newborn mice were hybridized with 2S Faza rat hepatoma cells and the hybrid cell colonies were analyzed for TAT activity, The results show the presence of inducible mouse TAT activity in mutant-2S Faza hybrid cells, thereby excluding the possibility that the structural gene for TAT is included in the gene sequences deleted in the mutants. Furthermore, determinations of mouse glucose-6-phosphate isomerase 1 as a marker eliminate chromosome 7 as the possible carrier of the TAT structural gene, which therefore appears to map on a different chromosome. The deletions interfering with normal enzyme activities apparently include genes other than the respective structural genes, namely those with essential functions in controlling the expression of the differentiated state of the liver cell.

The genetics of blind--a lethal factor in mice.

The linkage relationships of the lethal gene blind (Bld) in the mouse were investigated. The gene is located on chromosome 15, between uw and bt. The distance uw--Bld is 28.2 +/- 5.1 cM: Bld--bt is 14.9 +/- 2.7 cM: and Bld--Ca is 24.6 +/- 2.6 cM. Incomplete penetrance and/or reduced viability of Bld/+ animals was found when blind animals were crossed to several strains, but the cross female DBA/2J X male blind yielded complete penetrance and specificity. Evidence is presented for the presence of X-linked modifiers affecting the penetrance and possible increased viability of Bld in strain DBA.