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Oxoguanine DNA glycosylase (OGG1) is a major base excision repair protein responsible for excision of the mutagenic 8-oxoguanosine (8-oxoG) lesions from the genome. Despite OGG1's importance, the moderate phenotype of Ogg1-null (Ogg1(-/-)) mice is not well understood. This study addresses a mechanism by which Ogg1(-/-) cells limit accumulation of 8-oxoG in their genome. Our data reveal that a subset of Ogg1(-/-) cells shows higher ROS levels ((H)ROS cells), while approximately 85% of Ogg1(-/-) cells exhibit physiological levels of ROS ((L)ROS cells). Ogg1(-/-) cells were sorted based on their DCF fluorescence intensity to obtain (L)ROS and (H)ROS cell cultures. (L)ROS cultures proliferated at a rate comparable to Ogg1(+/+) and gradually accumulated cells exhibiting increased ROS and 8-oxoG levels. (L)ROS cells show a 2.8-fold increase in 8-oxoG level vs. (H)ROS cells (7-27-fold). Mitochondria of (H)ROS cells released more H(2)O(2) than (L)ROS and Ogg1(+/+) cells and were eliminated by apoptotic-like processes. These findings suggest that in the absence of OGG1, a surveillance system is activated that removes cells with extreme 8-oxoG levels from Ogg1(-/-) cultures. Whether similar mechanisms exists in tissues of Ogg1(-/-) mice is the focus of future investigations.

Isolation of newly synthesized mercurated RNA transcripts by chromatography on sulfhydryl-agarose has recently been used to reduce contamination by endogenous RNA derived from the chromatin template. We show that substantial RNA aggregation occurs during standard isolation procedures, causing significant retention of endogenous (unmercurated) RNA on sulfhydryl-agarose. We describe methods to reduce substantially this problem and discuss the implications of our findings for interpretation of previous hybridization and transcription experiments.

Access to the detailed structure of the globin (and other) genes has taught us at least three valuable, but not necessarily expected, lessons regarding the structure and evolution of the genes. Foremost, at least in terms of its surprising nature, is the fact that many genes are interrupted, i.e., they contain discontinuous blocks of coding and noncoding information. No less surprising are the accompanying facts that chromosomal DNA changes by the movement and rearrangement of large pieces of DNA and that genetic loci are highly and unexpectedly complex, consisting of arrays of related genes and pseudo (or apparently nonfunctional) genes. Here we review some of the evidence upon which these conclusions rest, and we try to form a coherent picture of gene evolution. The evidence that we shall use is based on studies of the mouse globin. Evidence from various other genetic systems leads us to believe that these genes serve as an instructive general model rather than an idiosyncratic one.

Structural and functional analysis of the mouse alpha-globin and beta-globin genes reveals that the globin genes are encoded in discontinous bits of coding information and that each gene locus is much more complex than was originally supposed. Each seems to consist of an array of several authentic genes as well as several apparently inactive pseudogenes. Comparison of the sequences of some of these genes to one another indicates that chromosomal DNA is a dynamic structure. Flanking and intervening sequences change in two ways: quickly, by duplication and extensive insertions and deletions, and slowly, by point mutation. Active coding sequences are usually limited to the slower mode of evolution. In addition to identifying fast and slow modes of evolution, it has also been possible to test the function of several signals that surround these genes and to identify those that appear to play a role in gene expression.

We describe the first polyA-containing processed pseudogene reported in the chicken. It includes a 0.52 kb open reading frame which could encode a 175 amino acid protein. The putative protein shows extensive homology to the ras oncogene superfamily, being most closely related to the yeast protein YP2. It is one of the two most divergent members of the ras superfamily yet described and is the most homologous of any ras-related protein to the G-protein alpha-transducin. The chicken genome contains at least one other gene highly homologous to CPS1; at least one member of the CPS1 family is active, but only early in chicken development. This pattern of expression, and the presence of mutations in regions known to activate human c-ras genes to oncogenicity, suggest that CPS1 may represent a new oncogene family.

The globin genes represent a complex set of sequences that are expressed in a coordinate fashion during the development of red blood cells. while this complex family of genes may consist of as many as ten to fourteen members [34], three of these genes have now been cloned and their entire nucleotide sequence determined. As was initially observed in the case of beta globin major gene, all are encoded in three distinct coding blocks separated by two intervening sequences of DNA. Their intervening sequences of DNA are preserved, with respect to location, but are widely divergent, with respect to size and sequence. The divided information in each gene is edited and spliced together at the level of its initial RNA transcript which is complementary to the entire gene sequence including its intervening sequences. Structural correlation analyses have allowed us to identify sites in all three genes that might be responsible for the initiation of transcription, RNA splicing, and poly A addition. The function of these sites has been tested by cloning these genes in an animal virus vector SV40. Such animal virus hybrids have been used to infect tissue culture cells and have directed the synthesis of both alpha and beta mouse globin in cells of monkey origin. These studies indicate that such signals operate across species barriers and further indicate that the animal virus vector system will be useful in elucidating their function.

An inverse relationship between a eukaryotic gene's level of methylation and its level of expression has long been recognized, and generally believed to result from reduced binding of postulated activator proteins to methylated target DNAs. There are, however, some genes where there is no apparent correlation between levels of methylation and gene expression, and even a small class where gene activation is correlated with increased methylation of the DNA. I propose a unifying hypothesis to explain these apparently divergent cases: methylation acts to reduce or abolish binding of regulatory proteins to their DNA target sites. In the majority of genes, methylation acts to block binding of activating factors; "indifferent" genes lack such methylation sites, while the minority class, which is more active when methylated, contains methylation sites which block binding of repressor proteins.

We have determined the nucleotide sequence of an embryonic beta-globin gene of the Balb/c mouse. It possesses structural features typical of known expressed beta-globins, including 5'-untranslated region, potential capping site, initiation and terminator codons, poly(A) addition signal, splicing signals, and intervening sequences. There is a striking 15-base homology between the putative RNA polymerase binding site of this gene and the corresponding region of a human embryonic beta-gene which may be important in the control of expression during embryonic development. The sequence of the gene predicts the sequence of the entire y2 protein, which has not previously been available. As expected from evolutionary considerations, it is more homologous to human embryonic beta-globin than to mouse adult beta-globin.

Hg-UMP-containing transcripts made from chick erythroid chromatins with E. coli RNA polymerase hybridize to chick globin cDNA. Contamination with endogenous globin RNA has been largely removed by purification on SH-agarose columns at 55 degrees C. Some endogenous globin mRNA sequences remain, probably as hybrids with "anti-sense" Hg-transcripts produced by RNA-dependent RNA synthesis. Heating to 115 degrees C before SH-agarose chromatography eliminates these contaminants. Hg-transcripts from adult and embryonic erythroid chromatins purified by this method are hybridized to globin cDNA; they contain a 4- to 6-fold higher proportion of globin-specific sequences (10-13 PPM) than do transcripts from brain chromatin. Dissociation of erythroid chromatins in salt and urea, followed by reconstitution using standard methods, destroys even this low degree of specificity.

The ras oncogenes play a crucial role in the regulation of cellular proliferation, probably by coupling growth factor receptors to the PI second messenger pathway. Additional experiments are needed to verify and detail this mechanism, and to examine the question of differing roles for the three ras variants.

We have determined the entire nucleotide sequence of a cloned mouse beta--globinminor gene and compared it to the closely related sequence of the betamajor gene. These two genes differ by nine amino acids and presumably evolved from a common ancestral gene as recently as 50 million years ago. Since these genes are closely linked and coordinately expressed, they provide an especially favorable opportunity to assess selection and mutation as these processes affect genes under similar constraints. We find that evolution has preserved these two genes in two short segments of DNA which include their immediately adjacent flanking regions. These regions presumably encode functions that are necessary for proper globin gene expression. In contrast, the more distal flanking sequences and major segments of the long intervening sequences have diverged much more sharply. The homology pattern in these genes also provides considerable insight into the mechanisms by which less constrained nucleotide sequences diverge rapidly. Change in such regions apparently occurs less by point mutation than by insertion, deletion and duplication of relatively short segments of the genome.