Barry Milavetz

Genetic Evidence for the Expression of ATP- and GTP-specific Succinyl-CoA Synthetases in Multicellular Eucaryotes

Highly ATP- and GTP-specific isoforms of succinyl-CoA synthetase in pigeon incorporate the same α-subunit, but different β-subunits (Johnson, J. D., Muhonen, W. W., and Lambeth, D. O. (1998) J. Biol. Chem. 273, 27573–27579). The sequences of the mature subunits were determined by methods based on reverse transcription-polymerase chain reaction. The 306-residue mature α-subunit in pigeon shows >88% identity to its homologues in pig and rat. The sequences of the mature ATP- and GTP-specific β-subunits (A-β and G-β, respectively) in pigeon are 54% identical. These sequences were used to identify expressed sequence tags for human and mouse that were highly homologous to G-β and A-β, respectively. The sequences for mature A-β and G-β in mouse and human were completed and verified by polymerase chain reaction. The sequence of A-β in pig was also obtained. The mammalian A-β sequences show >89% identity to each other; the G-β sequences are similarly related. However, pairwise comparisons of the A-β and G-β sequences revealed <53% identity. Alignment with two sequences of the β-subunit in Caenorhabditis elegans suggests that the A-β and G-β genes arose by duplication early in the evolution of multicellular eucaryotes. The expression of A-β is strong in numerous mouse and human tissues, which suggests that ATP-specific succinyl-CoA synthetase also plays an important role in species throughout the animal kingdom.

Decoding the histone H4 lysine 20 methylation mark

The molecular biology of histone H4 lysine 20 (H4K20) methylation, like many other post-translational modifications of histones, has been the subject of intensive interest in recent years. While there is an emerging consensus linking H4K20me1, H4K20me2, and H4K20me3 to transcription, repair, and constitutive heterochromatin, respectively, the specific details of these associations and the biological mechanisms by which the methylated histones are introduced and function are now the subject of active investigation. Although a large number of methylases capable of methylating H4K20 have been identified and characterized; there is no known demethylase of H4K20, though the search is ongoing. Additionally, many recent studies have been directed at understanding the role of methylated H4K20 and other histone modifications associated with different biological processes in the context of a combinatorial histone code. It seems likely that continued study of the methylation of H4K20 will yield extremely valuable insights concerning the regulation of histone modifications before and during cell division and the impact of these modifications on subsequent gene expression.

Characterization and Cloning of a Nucleoside-diphosphate Kinase Targeted to Matrix of Mitochondria in Pigeon

Nucleoside-diphosphate kinase (NDP kinase) from the matrix space of mitochondria in pigeon liver was purified to homogeneity. Degenerate oligonucleotide primers to the N-terminal sequence of the purified protein and the region containing the active site histidine were used in reverse transcriptase-polymerase chain reaction to obtain a major portion of the coding sequence for the mature protein. The sequences of the C and N termini of the mature protein, and eight residues in the signal peptide, were obtained by rapid amplification of cDNA end procedures. The entire coding sequence of a cytosolic form of NDP kinase was also determined. Both isoforms, which share 53% sequence identity, possess the characteristically conserved regions of known NDP kinases. The mature mitochondrial NDP kinase protein migrates in molecular sieving columns with an apparent molecular mass of about 66 kDa. It shows very high thermal stability even though it lacks the proline residue in thekiller of prune loop, and the Tyr/Glu C termini that are important in stabilizing other NDP kinases. The affinity of the mitochondrial isoform for adenine and guanine nucleotides is much higher than for pyrimidine nucleotides, but the enzyme is especially susceptible to substrate inhibition by GDP. Semi-quantitative reverse transcriptase-polymerase chain reaction showed that the relative levels of expression of the mitochondrial isoform are liver > kidney ≫ heart = brain > breast muscle. The cytosolic isoform is strongly and approximately equally expressed in these same five tissues. This work is the first characterization of a NDP kinase isoform that is found in the matrix space of mitochondria.

Generation of a nucleosome-free promoter region in SV40 does not require T-Antigen binding to site I

The relationship between T-antigen interactions at Site I and the presence of a nucleosome-free promoter in SV40 chromatin was examined by analyzing chromatin from mutants defective for T-antigen interaction at site I (cs 1085, scs111, and tsA58) and their parental wild-type strains (776, SVS, and VA45-54, respectively). As judged by sensitivity to digestion with restriction endonucleases that recognize unique sequences within the promoter region (BglI, KpnI, and MspI), a nucleosome-free promoter was observed in a substantially larger proportion of chromosomes from the defective mutants than their wild-type parents. This result demonstrates that T-antigen binding to site I is not necessary for setting the early boundary of the nucleosome-free region, although it may function directly or indirectly in determining the proportion of chromosomes containing this feature.

An analysis system for DNA gel electrophoresis images based on automatic thresholding an enhancement

Gel electrophoresis, a widely used technique to separate DNA according to their size and weight, generates images that can be analyzed automatically. Manual or semiautomatic image processing presents a bottleneck for further development and leads to reproducibility issues. In this paper, we present a fully automated system with high accuracy for analyzing DNA and proteins. The proposed algorithm consists of four main steps: automatic thresholding, shifting, filtering, and data processing. Automatic thresholding, used to equalize the gray values of the gel electrophoresis image background, is one of the novel operations in this algorithm. Enhancement is also used to improve poor quality images that have faint DNA bands. Experimental results show that the proposed technique eliminates defects due to noise for average quality gel electrophoresis images, while it also improves the quality of poor images.

Virion-mediated transfer of SV40 epigenetic information

In eukaryotes, epigenetic information can be encoded in parental cells through modification of histones and subsequently passed on to daughter cells in a process known as transgenerational epigenetic regulation. Simian Virus 40 (SV40) is a well-characterized virus whose small circular DNA genome is organized into chromatin and, as a consequence, undergoes many of the same biological processes observed in cellular chromatin. In order to determine whether SV40 is capable of transgenerational epigenetic regulation, we have analyzed SV40 chromatin from minichromosomes and virions for the presence of modified histones using various ChIP techniques and correlated these modifications with specific biological effects on the SV40 life cycle. Our results demonstrate that, like its cellular counterpart, SV40 chromatin is capable of passing biologically relevant transgenerational epigenetic information between infections.

HDAC inhibitors stimulate viral transcription by multiple mechanisms

BackgroundThe effects of histone deacetylase inhibitor (HDACi) treatment on SV40 transcription and replication were determined by monitoring the levels of early and late expression, the extent of replication, and the percentage of SV40 minichromosomes capable of transcription and replication following treatment with sodium butyrate (NaBu) and trichostatin A (TSA).ResultsThe HDACi treatment was found to maximally stimulate early transcription at early times and late transcription at late times through increased numbers of minichromosomes which carry RNA polymerase II (RNAPII) transcription complexes and increased occupancy of the transcribing minichromosomes by RNAPII. HDACi treatment also partially relieved the normal down-regulation of early transcription by T-antigen seen later in infection. The increased recruitment of transcribing minichromosomes at late times was correlated to a corresponding reduction in SV40 replication and the percentage of minichromosomes capable of replication.ConclusionThese results suggest that histone deacetylation plays a critical role in the regulation of many aspects of an SV40 lytic infection.

Transcriptional repression is epigenetically marked by H3K9 methylation during SV40 replication

BackgroundWe have recently shown that T-antigen binding to Site I results in the replication-dependent introduction of H3K9me1 into SV40 chromatin late in infection. Since H3K9me2 and H3K9me3 are also present late in infection, we determined whether their presence was also related to the status of ongoing transcription and replication. Transcription was either inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) or stimulated with sodium butyrate and the effects on histone modifications early and late in infection determined. The role of DNA replication was determined by concomitant inhibition of replication with aphidicolin.ResultsWe observed that H3K9me2/me3 was specifically introduced when transcription was inhibited during active replication. The introduction of H3K9me2/me3 that occurred when transcription was inhibited was partially blocked when replication was also inhibited. The introduction of H3K9me2/me3 did not require the presence of H3K9me1 since similar results were obtained with the mutant cs1085 whose chromatin contains very little H3K9me1.ConclusionsOur data suggest that methylation of H3K9 can occur either as a consequence of a specific repressive event such as T-antigen binding to Site I or as a result of a general repression of transcription in the presence of active replication. The results suggest that the nonproductive generation of transcription complexes as occurs following DRB treatment may be recognized by a ‘proof reading’ mechanism, which leads to the specific introduction of H3K9me2 and H3K9me3.

Transcription and replication result in distinct epigenetic marks following repression of early gene expression

Simian virus 40 (SV40) early transcription is repressed when the product of early transcription, T-antigen, binds to its cognate regulatory sequence, Site I, in the promoter of the SV40 minichromosome. Because SV40 minichromosomes undergo replication and transcription potentially repression could occur during active transcription or during DNA replication. Since repression is frequently epigenetically marked by the introduction of specific forms of methylated histone H3, we characterized the methylation of H3 tails during transcription and replication in wild-type SV40 minichromosomes and mutant minichromosomes which did not repress T-antigen expression. While repressed minichromosomes following replication were clearly marked with H3K9me1 and H3K4me1, minichromosomes repressed during early transcription were not similarly marked. Instead repression of early transcription was marked by a significant reduction in the level of H3K9me2. The replication dependent introduction of H3K9me1 and H3K4me1 into wild-type SV40 minichromosomes was also observed when replication was inhibited with aphidicolin. The results indicate that the histone modifications associated with repression can differ significantly depending upon whether the chromatin being repressed is undergoing transcription or replication.

Histone H4 lysine 20 mono- and tri-methylation define distinct biological processes in SV40 minichromosomes

The methylation profile of histone H4 on lysine 20 in SV40 chromatin during an infection was investigated using ChIP analyses with antibodies to monomethyl (H4K20me1), dimethyl (H4K20me2), and trimethyl (H4K20me3) histone H4. H4K20me1 was found in late-transcribing, uncoating, encapsidating, and replicating minichromosomes as well as in the SV40 chromatin present in virions. Its prevalence was greatest in virions and least in minichromosomes present between 4 and 24 hours post-infection. In contrast, H4K20me2 did not appear to be present and H4K20me3 appeared to be present only in minichromosomes obtained 30 minutes post-infection. The presence of H4K20me1 late in infection in replicating minichromosomes and its relative enrichment in virions suggested that it played a role in the encapsidation process. In contrast, the presence of H4K20me3 at the earliest stages of the infection and its subsequent relatively rapid loss along with SV40 chromatin suggested that it was functioning during the uncoating process.