Differential DNase I sensitivity of the albumin and alpha-fetoprotein genes in chromatin from rat tissues and cell lines.

We have examined the DNase I sensitivity of the albumin and alpha-fetoprotein (AFP) genes in different rat tissues (adult liver and kidney) and cloned cell lines (hepatoma 7777-C8, JF1 fibroblasts), which show drastic differences in the level of expression of these two genes. This was done by studying the disappearance of defined restriction endonuclease fragments of these genes as a function of limited DNase I digestion. The sensitivity of these genes was compared to that of a gene not expressed in the hepatic cells and to that of a ubiquitously expressed gene. In nuclei from adult rat liver the albumin and AFP genes were preferentially degraded by the nucleolytic action of DNase I, whereas they were not in rat kidney nuclei. In the hepatoma cells the AFP gene was much more sensitive to DNase I digestion than the albumin gene; both genes were very resistant to DNase I action in fibroblastic nuclei. When analyzed in relation to the level of gene expression our results indicate that alterations in the chromatin structure of the albumin and AFP genes might be involved in the early establishment of the tissue-specific potential of overt gene expression; such alterations reflected in an altered DNase I sensitivity do not appear to be responsible for the changes in gene activity occurring during the terminal differentiation of the hepatocyte; and modifications in the chromatin structure of these genes might occur during oncogenic events; these structural modifications could be related to the changes in gene expression observed in hepatocarcinogenic processes.     

Native genomic blotting: high-resolution mapping of DNase I-hypersensitive sites and protein-DNA interactions.

DNase I-hypersensitive sites are observed in the promoter regions of actively expressed genes, potentially active genes, and genes that were once active. We have developed an approach that greatly increases the resolution for mapping these sites by electrophoresing genomic DNA on native polyacrylamide gels prior to electroblotting and hybridization. This improved method has been used to scan the promoter and coding region of a cell-cycle-dependent human histone H4 gene with an accuracy of +/-5-10 base pairs. Protein-DNA interactions can be seen in the autoradiograph as light areas and DNase I-hypersensitive sites as dark bands. Therefore, this method provides a rapid and relatively simple means to accurately localize protein-DNA interactions as well as DNase I-hypersensitive sites, thus directly displaying DNase I hypersensitivity and protein-DNA complexes on one autoradiograph. It also potentially allows the analysis of small changes in DNase I-hypersensitive sites under various biological conditions. With this technique rather large regions of DNA can be screened to determine areas that should be analyzed by more sophisticated methods, such as genomic sequencing or gel retardation assays.     

Mapping of DNase I-hypersensitive sites in the upstream DNA of human embryonic epsilon-globin gene in K562 leukemia cells.

We have mapped the DNase I-hypersensitive sites around the epsilon-globin and c-myc genes in two human leukemia cell lines K562 and HL60. In K562 cells in which the epsilon-globin gene is transcribed, six DNase I-hypersensitive sites are found in 6 kilobases (kb) of upstream flanking DNA; in HL60 cells in which the c-myc gene is expressed, two DNase I-hypersensitive sites are observed in 2 kb of upstream DNA. Neither the inactive epsilon-globin gene in HL60 cells nor the inactive c-myc gene in K562 cells displays such upstream DNase I-hypersensitive sites. Our results are consistent with previous studies that have shown DNase I-hypersensitive sites within 1 kb of the 5' end of other expressed genes. In addition, we have found sites displaying even more DNase I sensitivity further upstream of expressed epsilon-globin and c-myc genes. Among the six DNase I-hypersensitive sites of the expressed epsilon-globin gene in K562 cells, the most sensitive site is located about 6 kb upstream of the epsilon-globin gene. When correlated with the DNA sequence upstream of the epsilon-globin gene, this site was found to correspond to a region that contains a stretch of 28 consecutive Ts, three enhancer core-like sequences, and a stretch of consecutive (C-A)15(T-A)6 alternating purine and pyrimidine bases. These findings suggest the possibility that an enhancer element for epsilon-globin gene expression resides within this DNase I-hypersensitive site.     

Dissection of the enhancer activity of beta-globin 5' DNase I-hypersensitive site 2 in transgenic mice.

The beta-globin locus control region (LCR) consists of four erythroid-specific DNase I-hypersensitive sites, which are necessary for high-level expression of the beta-like globin genes in erythroid tissues. One of these sites, designated 5'HS-2, functions as an erythroid-specific enhancer element in transfection and transgenic mouse experiments. Recent transfection experiments and studies of DNA-protein interactions have localized the 5'HS-2 enhancer to 18 nucleotides that contain a binding site for both the erythroid-specific factor nuclear factor erythroid 2 (NFE-2) and for activator protein 1 (AP-1). To define the sequences necessary for in vivo enhancer activity, several deletion mutants of 5'HS-2 were linked to the human beta-globin gene and their activity was tested in transgenic mice. Three upstream fragments of 5'HS-2 [341, 374, and 412 base pairs (bp)], each of which contained the NFE-2/AP-1 sequences, resulted in beta-globin expression at levels equivalent to or higher than those observed with the entire 732-bp 5'HS-2 fragment. In contrast, a 358-bp downstream portion of 5'HS-2, which lacked the NFE-2/AP-1 sequences, resulted in beta-globin expression at the low levels seen with the beta-globin gene alone. Removal of the NFE-2/AP-1 sequences by a 67-bp internal deletion resulted in similar low levels of beta-globin expression. A 100-bp 5' fragment that contained the NFE-2/AP-1 sequences resulted in beta-globin expression that was higher than the beta-globin gene alone but lower than the entire 5'HS-2 fragment or the three larger upstream fragments. These studies demonstrate that the NFE-2/AP-1 sequences are essential for enhancer activity of 5'HS-2 but that other sequences are required for full activity in vivo.     

Light-regulated changes in DNase I hypersensitive sites in the rRNA genes of Pisum sativum

We have examined the rDNA chromatin of Pisum sativum plants grown with or without exposure to light for the presence of DNase I hypersensitive sites and possible developmental changes in their distribution. Isolated nuclei from pea seedlings were incubated with various concentrations of DNase I. To visualize the hypersensitive sites, DNA purified from these nuclei was restricted and analyzed by gel blot hybridization. We find that several sites exist in both the coding and noncoding regions of rDNA repeating units. Several of the sites in the nontranscribed spacer region are present in the light but are absent in the dark. Conversely, the hypersensitive sites within the mature rRNA coding regions are present in the dark but absent in the light. There are two major length variants of the rRNA genes in P. sativum var. Alaska. The sites in the nontranscribed spacer region that appear during the light treatment occur only in the shorter of these two length variants in this cultivar.     

Detection of genes with a potential for suppressing the transformed phenotype associated with activated ras genes.

Seven morphologically nontransformed (flat) revertants with reduced tumorigenicity in vivo have been isolated from populations of Kirsten sarcoma virus-transformed NIH 3T3 cells transfected with a cDNA expression library of normal human fibroblasts. Each revertant harbors 1-10 recombinant plasmids per cell and retains a rescuable transforming virus as well as high level expression of v-Ki-ras-specific RNA and the viral oncogene product, p21v-Ki-ras. Transformed phenotypes are suppressed in cell hybrids generated by fusing each revertant to v-Ki-ras-transformed NIH 3T3 cells. From two of the revertant lines, plasmids capable of giving rise to flat secondary transfectants have been recovered. Thus, in some, if not all, of the revertants, transfected cDNAs seem to be responsible for the suppression of specific transformed phenotypes.     

alpha-Fetoprotein and albumin genes are in tandem in the mouse genome

The urine alpha-fetoprotein (AFP) and serum albumin genes most probably arose in evolution as the consequence of a duplication of a common ancestral gene. They have both been previously mapped to chromosome 5 in the mouse. We now have evidence that these genes are closely linked. By using a unique copy DNA probe derived from previously cloned AFP 5' flanking DNA, a recombinant DNA phage has been isolated, from a bacteriophage DNA library, that contains sequences flanking the 5' end of the AFP gene and the 3' end of the albumin gene. Restriction endonuclease mapping and DNA sequence determination of the recombinant phage and comparison to total genomic DNA confirmed that the genes are in tandem, 13.5 kilobase pairs apart, with the albumin gene to the 5' side of the AFP gene. Thus, they are transcribed from the same strand of DNA.     

Long-term maintenance of the adult pattern of liver-specific expression for P-450b, P-450e, albumin and alpha-fetoprotein genes in intrasplenically transplanted hepatocytes.

Hepatocytes isolated from livers of Fischer 344 rats and transplanted into the spleens of rats from the same strain survived for at least 15 mo in the absence of immunosuppressive drugs. Hepatocytes attached themselves only in the red pulp of the spleen, most commonly in clumps without a discernible structure. Throughout the 15-mo period, intrasplenically transplanted hepatocytes expressed cytochrome P-450b, P-450e and albumin messenger RNAs, whereas alpha-fetoprotein messenger RNA was not expressed. In addition, the relative expression of albumin and P-450 genes was similar to that in liver. For example, albumin messenger RNA was expressed to higher levels than P-450b or e messenger RNAs. Northern blots hybridized with oligonucleotides specific for P-450b or P-450e showed that, as in liver, both P-450b and P-450e genes were induced in response to phenobarbital. Quantitative slot-blot hybridizations performed at 15 days and 1, 6, and 15 mo after hepatocyte transplantation revealed that cytochrome P-450b and P-450e messenger RNAs were induced about 20- to 30-fold by a single dose of phenobarbital. This level of induction was also similar to that observed in liver. Hence, intrasplenically transplanted hepatocytes represent a unique system in which hepatocytes, cultured in an extrahepatic in vivo environment, maintain for at least 15 mo a pattern of expression for these four liver genes similar to that in the adult liver. Moreover, these studies suggest that neither the organization of liver into acini nor a specific zonal sinusoidal microenvironment is necessary for adult hepatocytes to respond to phenobarbital with induction of P-450b and P-450e genes.     

Endogenous viral genes of the White Leghorn chicken: common site of residence and sites associated with specific phenotypes of viral gene expression.

The DNAs from greater than 150 individual White Leghorn chickens were digested with restriction endonucleases BamHI, EcoRI, HindIII, and Sst I, fractionated by gel electrophoresis, denatured, and transferred to nitrocellulose filters. Fragments containing the endogenous viral genes were detected by hybridization with 70S Rous associated virus type 2[32P]RNA. Embryos of several different phenotypes with respect to production of the endogenous virus and expression of viral group-specific antigen and viral envelope protein were analyzed. DNA from birds of each phenotype produced a distinctive pattern of fragments containing viral genetic information. Individual fragments were seen to segregate as genetic loci in mating experiments. From the fragment patterns and the segregation data, the following conclusions were drawn with respect to the sites of residence in the chicken chromosome of the endogenous viral genes: (i) the DNA of all chickens contains viral genetic information in at least one site, and this site of residence appears to be the same in all chickens analyzed; (ii) four other sites have been identified, and the presence of viral information at each of these sites is always accompanied by a specific phenotype of endogenous viral gene expression; (iii) in addition to the above-mentioned five sites, a small number of other sites have been identified which are not associated with a known phenotype.