W. D. Park

In vitro synthesis of DNA complementary to polyadenylated histone messenger RNA

Summary The messenger RNAs for histone polypeptides were isolated from S phase HeLa S3 cells and adenylic acid residues were enzymatically added to the 3′-OH end of the molecules with a eukaryotic ATP: polynucleotidylexotransferase. The polyadenylated histone messenger RNAs were then used as templates for the synthesis of complementary DNAs by RNA-dependent DNA polymerase from Rous sarcoma virus.

Cell cycle stage-specific transcription of histone genes.

Abstract DNA complementary to histone messenger RNAs was utilized to assay the in vitro transcripts from chromatin of G1 and S phase HeLa S3 cells for the presence of histone specific sequences. The complementary DNA was prepared by transcribing, with an RNA-dependent DNA polymerase, histone messenger RNAs isolated from the polyribosomes of S phase HeLa S3 cells to which adenylic acid residues had been enzymatically added. While the RNA transcripts of S phase chromatin contained histone specific sequences, such sequences were not detected in the RNA transcripts from G1 chromatin. These results suggest that transcription of the genes for histone polypeptides is restricted to the S phase of the cell cycle.

Evidence that the coupling of histone gene expression and DNA synthesis in HeLa S3 cells is not mediated at the transcriptional level

Summary Representation of histone mRNA sequences in various intracellular fractions and in vitro transcripts of chromatin was examined after inhibition of DNA synthesis in S phase HeLa S 3 cells by cytosine arabinoside or hydroxyurea. Histone mRNA sequences were assayed by hybridization to a 3 H-labeled, single-stranded DNA complementary to histone mRNAs. Both inhibitors bring about a drastic reduction (greater than 99%) in the level of histone mRNA sequences on polysomes. The representation of histone mRNA sequences in nuclei and in chromatin transcripts is not affected by treatment for 30 min with cytosine arabinoside or hydroxyurea. Cytosine arabinoside or hydroxyurea treatment results in an elevated level of histone mRNA sequences in the post-polysomal cytoplasmic fraction. Taken together these results provide evidence that in vivo as well as in vitro coupling of histone gene expression and DNA synthesis is not mediated at the transcriptional level. The specific post-transcriptional process at which the coupling mechanism is operative remains to be identified.

Nonhistone Chromosomal Proteins and Histone Gene Transcription

Publisher Summary This chapter discusses that throughout the cell cycle of continuously dividing cells, as well as after the stimulation of nondividing cells to proliferate, a complex and interdependent series of biochemical events occur requiring modifications in the expression of information encoded in the genome. Hence, the cell cycle provides an effective model system for studying the regulation of gene readout. For the past several years, the laboratory has been focusing on the cell-cycle, stage-specific regulation of a defined set of genetic sequences—that coding for the histones. The chapter reviews that in continuously dividing cells as well as after stimulation of nondividing cells to proliferate, (1) regulation of histone gene expression resides, at least in part, at the transcriptional level, and (2) a subset of the nonhistone chromosomal proteins associated with the genome during the S-phase of the cell cycle is responsible for activation of histone gene transcription when DNA replication occurs.

Activation of histone gene transcription from chromatin of G1 hela cells by s-phase non-histone chromosomal proteins

Non-histone chromosomal proteins have been implicated as having an important function in the regulation of gene expression [l-9] . Specifically, these proteins have been shown to be involved in controlling the transcription of globin genes in erythroid cells [ 1 O-l 2] and histone genes during the cell cycle [ 13-141. In HeLa cells, synthesis of histones is restricted to the period of the cell cycle during which DNA is replicated (S-phase) [15-l 81, and translation [15-17, 19-211 as well as hybridization data [22] indicate that histone mRNAs are associated with polysomes only at this time. Using a 3H-labeled single stranded complementary DNA as a probe for histone mRNA sequences [23], we have recently demonstrated that histone genes are transcribed in vitro from chromatin isolated from S-phase cells, but that chromatin isolated from Gr phase cells does not serve as a template for the transcription of histone sequences [24]. These results suggest that histone genes are transiently expressed during the period of DNA replication and that regulation of histone gene expression occurs at least in partat the transcriptional level. We have shown through chromatin in reconstitution studies that the non-histone chromosomal protein component of the genome is responsible for the difference in the in vitro transcription of histone sequences from Gi and S-phase chromatin [ 131. The present study demonstrates that when chromatin from Gr phase HeLa cells is reconstituted in the presence of nonhistone chromosomal proteins isolated from S-phase cells, a dose dependent activation of histone gene transcription is observed.

Fractionation of nonhistone chromosomal proteins.

Publisher Summary This chapter describes the methods for the fractionation of nonhistone chromosomal proteins. The chapter focuses on preparative-scale methods for the fractionation of nonhistone chromosomal proteins. The fractionation approaches are surveyed, and then QAE-Sephadex chromatography of nonhistone chromosomal proteins is focused on. One approach to fractionation of nonhistone chromosomal proteins has been to extract classes of these molecules with various concentrations of salt and urea—the urea being used to facilitate extraction of tenaciously bound molecules and to maintain extracted proteins in a soluble state. The nonhistone chromosomal proteins can be isolated as a total class of macromolecules and then fractionated. Such procedures require separation of DNA from the total chromosomal proteins as an initial step, followed by fractionation of the total chromosomal proteins into histones and nonhistone chromosomal proteins. The method used for fractionation of nonhistone chromosomal proteins is the ion-exchange chromatography on QAE-Sephadex. The objective of nonhistone chromosomal protein fractionation is to isolate classes, and individual species that are directly involved in the regulation of specific genetic sequences.