Adult White Leghorn chickens were rendered anemic by injection with 1-acetyl-2-phenylhydrazine and then treated with parenteral 5-azacytidine, and levels of embryonic globin RNA in circulating reticulocytes were measured. A very small but detectable amount of correctly initiated embryonic p-type globin RNA was detected in reticulocytes from birds treated with 5-azacytidine, while none was detected in reticulocytes from those receiving only phenylhydrazine or phenylhydrazine plus 1-f3-D-arabinofuranosylcytosine (cytosine arabinonucleoside). An attempt to increase embryonic globin RNA induction by treatment with parenteral sodium butyrate after 7 days of 5-azacytidine administration resulted in a 5-to 10-fold increase in the level of embryonic globin RNA. However, sodium butyrate did not induce embryonic gene expression when given alone or after treatment with cytosine arabinonucleoside. Sodium butyrate treatment also caused a DNase I-hypersensitive site to be exposed at the 5' end of the p-globin gene only after 5-azacytidine induced demethylation of several CpG sites in and around the gene. (20). In these latter studies there remains some question as to whether increased t-globin gene expression is due to gene demethylation, cell selection, or both, since 5-azacytidine is known to have a variety of toxic effects on cells, and since y-globin expression can be stimulated in adults by several types of bone marrow stress.As part of ongoing studies aimed at elucidating mechanisms that regulate globin gene expression, we have attempted to study the effects of demethylation of embryonic globin genes in anemic adult chickens. We report here that while 5-azacytidine treatment causes nearly complete demethylation of the p embryonic globin gene in adult erythroid cells, only a minimal amount of p-globin RNA is detectable, and the surrounding chromatin structure, as assayed by DNase I digestion, does not differ from untreated controls. On the other hand, pharmacologic doses of sodium butyrate greatly increase the amount of embryonic p-globin RNA in adult reticulocytes and result in the exposure of a 5' DNase I-hypersensitive site in some of the reticulocyte nuclei, but only when the gene has first become demethylated by 5-azacytidine treatment. MATERIALS AND METHODS Treatment of Animals and Blood Collection. Adult WhiteLeghorn hens (Yoder, Kalona, IA) were rendered anemic by five daily intramuscular injections with 1-acetyl-2-phenylhydrazine (Sigma) at 20 mg/kg or by phlebotomy of 10 ml of whole blood per day to achieve a hematocrit of 18-22% (normal 35-40%). Reticulocyte 3954The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

No abstract

An optional 46-base-pair G+C-rich element (GC cluster) in the coding region of the yeast mitochondrial varl gene inserts preferentially in crosses into recipient alleles that lack the sequence. Unlike a similar gene conversion event involving the insertion of an optional 1143-base-pair intron, the mitochondrial 21S rRNA gene, which requires the action of a protein encoded by a gene within that intron, conversion of the var) GC cluster does not require any protein product of the mitochondrial genome. We have detected double-strand breaks in the varl gene in mitochondrial DNA isolated from unmated haploid p+ and p-strains at or near the boundaries of the optional GC cluster, as well as at a conserved copy of that sequence 160 base pairs upstream. No double-strand breaks were detected in the recipient varl DNA molecules in the vicinity of the optional GC cluster target sequence. This contrasts with 21S rRNA-encoding DNA (rDNA) intron conversion where the recipient, but not the donor DNA, is cleaved at the element insertion site. These results suggest that although the 21S rDNA intron and the var) GC duster are preferentially inserted into their respective short alleles, these conversions probably occur by different mechanisms.The -80-kilobase (kb) mitochondrial genome of Saccharomyces cerevisiae contains two distinct classes of optional DNA sequences. One class is evident among yeast strains as a variable number of introns located within the genes encoding cytochrome b, cytochrome oxidase subunit I, and the large (21S) rRNA (1); the other class is made up of a large number (>150) of G+C-rich sequences, called GC clusters, most of which are 30-60 base pairs (bp) long and are found dispersed throughout the yeast mitochondrial genome, mostly in the intergenic spaces (2-4). These GC clusters have been subdivided into various groups based on primary sequence homologies (4), and except for those few GC clusters located within the coding sequences of genes, their functions are unknown.For two particular optional mtDNA sequences, specialized recombination is evident in which an allele lacking that sequence acquires it in crosses by unidirectional gene conversion (5). One such recombination involves an optional 1143-bp intron, called co, located within the 21S rRNA gene.In crosses between wt and w-strains, nearly all of the calleles are converted to w+ (6, 7). The mechanism of this recombination event is now partly understood and closely resembles mating type interconversion in yeast nuclear DNA (8). Intron insertion is mediated in part by a protein, called fiti, encoded entirely within the 1143-bp intron (9, 10).That protein is a sequence-specific endonuclease that catalyzes a double-strand break in the DNA of the w-allele at the site of intron insertion (11); conversion probably occurs as a result of the replicative repair of the double-strand break in which the DNA of the a+ allele is used as a template (6-9). This conversion event thus has a clear dependence on mitochondrial protein synthesis.The other specializ...

The signal pathways by which interferon-gamma (IFN-gamma) is able to up-regulate major histocompatibility complex (MHC) class I transcription were studied in two human hematopoietic tumor cell lines, K562 and Ramos. These studies suggest that the IFN-gamma signal is transduced via an H7- and staurosporine-sensitive kinase that is distinct from protein kinase C (PKC) and protein kinase A (PKA) in both cell types. Ramos cells appear to utilize an additional pathway involving double-stranded RNA-dependent protein kinase. PKC and possibly PKA appear to be involved in one or more intersecting pathways by which agonists of these kinases are able to act synergistically with IFN-gamma, but activation of these latter pathways is neither necessary nor sufficient for induction of MHC class I transcription. Modulation of G-protein- and Ca2+-calmodulin-associated pathways and arachidonic acid metabolism had no effect on constitutive or IFN-gamma-stimulated class I transcription. The class I stimulatory factor produced in response to IFN-gamma treatment appears to have a short t1/2. The identity of this factor is unknown, but is likely to be distinct from known mediators of IFN-stimulated transcription. Gene and cell-type specificity in the signal transduction pathways utilized by IFN-gamma implies that such pathways may be useful targets for experimental and therapeutic manipulation.