GTPase RhoB: an early predictor of neuronal death after transient focal ischemia in mice.

Applying the recently developed DNA array technique to a murine stroke model, we found that the gene coding for RhoB, a member of the family of GTPases that regulate a variety of signal transduction pathways, is upregulated in ischemia-damaged neurons. RhoB immunoreactivity precedes DNA single-strand breaks and heralds the evolving infarct, making it an early predictor of neuronal death. Expression of RhoB colocalized with drastic rearrangement of the actin cytoarchitecture indicates a role for Rho in postischemic morphological changes. Apoptosis in a murine hippocampal cell line was also associated with an early increase in RhoB protein. Activation of caspase-3, a crucial step in apoptosis, could be inhibited by cytochalasin D, a substance that counteracts the actin-modulating activity of Rho GTPases, indicating that Rho proteins may have impact on injury-initiated neuronal signal transduction. Our findings make Rho GTPases potential targets for the development of drugs aimed at limiting neuronal death following brain damage.

Adenovirus type 12 DNA firmly associates with mammalian chromosomes early after virus infection or after DNA transfer by the addition of DNA to the cell culture medium.

Human adenovirus type 12 (Ad12) infects human cells productively and leads to viral replication, whereas infection of hamster cells remains abortive, with total blocks in viral DNA replication and late viral gene transcription. The intranuclear fate of Ad12 DNA in productively infected human cells and in abortively infected hamster cells was monitored by using the fluorescent in situ hybridization (FISH) technique. Human HeLa cells, primary human umbilical cord fibroblasts, hamster BHK21 cells, primary embryonal hamster cells, and the Ad12-transformed T637 hamster cell line were studied. As early as 2 h after infection, extensive association of Ad12 DNA with metaphase chromosomes was demonstrated by FISH in all of these cells. Chromosomal association continued until late (24 to 28 h) after infection, when about 100% of the human cell nuclei and 70 to 80% of the hamster cell nuclei showed distinct FISH signals. This chromosomal association of Ad12 DNA in infected cells seemed to be rather firm, since it proved to be resistant to mechanically stretching the chromosomes and to different types of chemical treatment. Moreover, laser scan microscopy of mechanically stretched chromosomes from Ad12-infected HeLa cells and from the Ad12-transformed T637 cell line, with about 20 copies of Ad12 DNA provably integrated, revealed identical FISH patterns. Therefore, it was likely that even in infected cells the chromosomal association of Ad12 DNA was very similar to the integrated state. Late in productively infected cells, large nuclear areas were taken over by viral DNA replication, as visualized by FISH in interphase nuclei. Chromosomal association at many sites was frequently limited to one chromatid, but signals in adjacent positions on both chromatids were also seen. Upon the long-term cultivation and passage of abortively infected BHK21 cells for 96 h after infection, a gradual decrease of viral DNA association with chromosomes was observed. Integration of Ad12 DNA in hamster cells early after infection was previously documented, and recombination between viral and cellular DNAs in human cells was also shown. The FISH data on extensive chromosomal association of Ad12 DNA suggest a means to study the pathway of Ad12 DNA from early steps in viral infection via chromosomal interactions to integration events. In a different approach, Ad12 DNA, Ad12 DNA with the terminal protein covalently linked to its ends (Ad12 DNA-TP), or Ad2 DNA was simply added to the culture medium of HeLa or BHK21 cells. Precipitation or selection procedures were avoided. Depending on the experimental conditions, up to 25 to 30% of the interphase nuclei of HeLa cells and 9 to 19% of the interphase nuclei of BHK21 cells showed positive FISH signals at 24 h after the addition of DNA. Viral DNA also became associated in some cases with both chromatids. The uptake of Ad12 DNA-TP appeared to be 10 to 20 times more efficient than that of Ad12 DNA completely freed of proteins. Control bacteriophage lambda, M13, or plasmid DNA could not be detected in the nuclei under these conditions.

A fully 5'-CG-3' but not a 5'-CCGG-3' methylated late frog virus 3 promoter retains activity.

Several lines of evidence demonstrate that the DNA of the iridovirus frog virus 3 (FV3) is methylated in all 5'-CG-3' sequences both in virion DNA and in the intracellular viral DNA at late times after infection. The 5-methyldeoxycytidine residues in this viral DNA occur exclusively in 5'-CG-3' dinucleotide positions. We have cloned and determined the nucleotide sequence of the L1140 gene and its promoter from FV3 DNA. The gene encodes a 40-kDa protein. The results of transcriptional pattern analyses for this gene in fathead minnow fish cells document that this gene is transcribed exclusively late after FV3 infection. The L1140 gene and its promoter are fully methylated at late times after infection. We have been interested in resolving the apparent paradox that the methylated L1140 promoter is methylated and active late in FV3-infected cells. Of course, the possibility cannot be excluded that one or a few 5'-CG-3' sequences outside restriction endonuclease sites escaped de novo methylation after FV3 DNA replication. We have devised a construct that places the chloramphenicol acetyltransferase gene under the control of the L1140 promoter. Upon transfection, this construct exhibits activity only in FV3-infected BHK-21 hamster cells, not in uninfected BHK-21 cells. The fully 5'-CG-3' or 5'-GCGC-3' (HhaI) methylated, HpaII-mock-methylated, or unmethylated L1140 promoter-chloramphenicol acetyltransferase gene construct is active in FV3-infected BHK-21 cells, whereas the same construct 5'-CCGG-3' (HpaII) methylated has lost activity. Apparently, complete methylation of the late L1140 promoter in FV3 DNA is compatible with activity. However, a very specific 5'-CCGG-3' methylation pattern that does not naturally occur in authentic FV3 DNA in infected cells abrogates promoter function. These results further support the notion that very specific patterns of methylation are required to inhibit or inactivate viral promoters.

Patterns of frog virus 3 DNA methylation and DNA methyltransferase activity in nuclei of infected cells.

The iridovirus frog virus 3 (FV3) can replicate in culture in fat head minnow (FHM) fish cells or in BHK-21 hamster cells. Viral DNA replication commences about 3 h after infection of FHM cells with FV3. Between 3 and 6 h postinfection (p.i.), a portion of the intranuclear FV3 DNA is partly unmethylated. At later times, p.i., all of the viral DNA in the nuclear and cytoplasmic compartments is methylated at the 5'-CCGG-3' sequences. Cytoplasmic FV3 DNA has not been found unmethylated. We have cloned viral DNA fragments from methylated virion DNA. By using the genomic sequencing technique, it has been demonstrated for segments of the FV3 DNA replicated both in FHM fish and BHK21 hamster cells that in a stretch encompassing a total of 350 bp, all of the analyzed 5'-CG-3' dinucleotides are methylated. The modified nucleotide 5-methyldeoxycytidine is present exclusively in the 5'-CG-3' dinucleotide combination. In the cloned FV3 DNA fragment p21A, an open reading frame has been located. The 5' region of this presumptive viral gene is also methylated in all 5'-CG-3' positions. DNA methyltransferase activity has been detected in the nuclei of FV3-infected FHM cells at 4, 11, and 20 h p.i. In the cytoplasmic fraction, comparable activity has not been observed. These data are consistent with the interpretation that FV3 DNA is newly synthesized and de novo methylated in the nuclei of infected FHM cells and subsequently exported into the cytoplasm for viral assembly.

Patterns of DNA methylation are indistinguishable in different individuals over a wide range of human DNA sequences.

Patterns of DNA methylation at 5'-CCGG-3' and 5'-GCGC-3' sequences were determined in about 570 kb, equivalent to about 0.02% of the human genome, by using HpaII and HhaI restriction endonucleases, respectively, and randomly selected cosmid clones of human DNA as hybridization probes. Many of these human DNA sequences were of the repetitive type. The DNAs from human lymphocytes, from a mixture of all blood cells or from several established human cell lines (HeLa, KB, 293, or DEV) were included in these analyses. In the segments of the human genome investigated, the patterns of DNA methylation were characterized by often completely or partly methylated 5'-CCGG-3' or by partly methylated 5'-GCGC-3' sequences. Even among individuals of different genetic origins (East-Asian or Caucasian), these patterns of DNA methylation proved indistinguishable by the method applied. The cytokine-dependent stimulation of human lymphocytes to replicate in culture did not affect the stability of these patterns. In the same DNA sequences from several human cell lines, much lower levels of DNA methylation were observed. In human cell lines some of the investigated sequences were unmethylated. The results presented lend credence to the notion that the human genome exhibits highly cell type-specific patterns of DNA methylation which are often indistinguishable among different individuals even of different genetic backgrounds.

Patterns of DNA methylation in selected human genes in different Hodgkin's lymphoma and leukemia cell lines and in normal human lymphocytes.

The human genome, like many other genomes, harbors highly specific patterns of DNA methylation which have not yet been systematically studied. In a limited investigation on the genes for tumor necrosis factors-alpha and -beta, a surprising interindividual concordance in the patterns of DNA methylation at the nucleotide level has been demonstrated earlier by using the genomic sequencing method on DNA from individuals of very different ethnic origins. Patterns of DNA methylation could perhaps serve as indicators for genetic activities. These activities would not have to be restricted to gene transcription but could relate to other genetic activities in the cell. DNA methylation patterns are known to be cell type-specific. We have now initiated a study of these DNA patterns in human lymphocytes and in human cell lines of different malignant origins. Several of the proto-oncogenes, parts of the genes for tumor necrosis factor-alpha and -beta, the insulin receptor and lamin C have been used as hybridization probes. We have relied to some extent on the documented observation that the methylation patterns at 5'-CCGG-3' (HpaII/MspI) sequences yield a reflection of patterns at all 5'-CG-3' sequences. Three main types of patterns have been observed. Some of the probed segments are completely unmethylated; others are fully methylated, most of the areas are partly methylated exhibiting complex patterns at the 5'-CCGG-3' sites. In different tumor cell lines, different DNA methylation patterns are apparent for the same DNA probes. Comparisons of the methylation patterns in a given DNA segment between DNA from primary normal human lymphocytes and DNA from different tumor cell lines reveal changes in these patterns in several instances.