Jostein Holtlund

On the presence of two new high mobility group-like proteins in HeLa S3 cells

Two phosphorylated HMG‐like proteins with M r ≈ 10 000 have been isolated from HeLa S3 cells, one being present in metaphase and one in interphase cells. The amino acid compositions of these proteins are very similar but differ from the known HMG proteins. However, they exhibit similarities being rich in proline, basic and acidic amino acids. A possible role in chromatin condensation of the HMG‐like protein characteristic for metaphase cells is suggested.

On the phosphorylation of low molecular mass HMG (high mobility group) proteins in Ehrlich ascites cells.

This paper shows that the low molecular mass HMG proteins 14 and 17 do not seem to be phosphorylated in Ehrlich ascites cells whereas two other small HMG proteins designated HMG I and Y are. Amino acid analysis and peptide mapping of all four proteins demonstrated that HMG I and Y were not phosphorylated modifications of HMG 14 or 17.

The human chromosomal protein HMG I contains two identical palindrome amino acid sequences

The sequence of 105 amino acids of the human high mobility group chromosomal protein HMG I has been determined. The most striking feature of this sequence is two identical palindrome sequences: pro-arg-gly-arg-pro, which together with a third related sequence: gly-arg-pro-arg, may represent the binding sites of HMG I to clusters of A-T base pairs in DNA.

HMG 17 in metaphase-arrested and interphase HeLa S3 cells

Proteins HMG 17 and 14 belong to a group of proteins designated high mobility group proteins [ 11. Experiments have indicated that transcribed genes are enriched in HMG 17 and 14 or the homologous protein H6 [2-51. The exact location of these proteins on the nucleosomes of transcribed sequences has not yet been settled. Certain experiments indicate that in the transcribed regions HMG 17 and 14 replace Hl , and it has been suggested that this results in a local unfolding of chromatin, thereby facilitating transcription [6]. On the other hand, these proteins may not only play a role during transcription since they remain attached to extensively digested nucleosomes [6] and reconstitution experiments [7,8] indicate that HMG 17 and 14 readily bind to the 145 base pair core particle at a stoichiometry of 1 and 2 molecules/core. 2.1. Propagation of HeLa S3 cells Interphase cells were grown in suspension culture as in [9]. To obtain cells arrested in metaphase, cells were grown in roller tubes and harvested after treatment with colcemid (0.05 pg/ml) as in [9]; 95% of the cells were in metaphase.

ON THE PRESENCE OF POLY~ADP-RIBOSE~ POLYMERASE ACTIVITY IN METAPHASE CHROMOSOMES FROM HeLa S3 CELLS

In a number of different eucaryotic cells, interphase chromatin contains poly(ADP-ribose) polymerase [l-3 J. It incorporates the ADP-ribose moiety of NAD into poly(ADP-ribose) of varying chainlength (l-40 residues). The polymer is covalently bound to histones and non&stone proteins. The polymerase has been found also in the cytoplasm of HeLa cells tightly bound to ribosomes [4]. This enzyme has also been detected in mitotic cells [5] but its location in isolated metaphase chromosomes not yet examined. Since the occurrence of this enzyme in chromatin from interphase cells does not necessarily mean that it is present in an enzymaticaIly active form in metaphase chromosomes, it was considered important to establish this. We show that metaphase chromosomes from HeLa S3 cells contain the enzyme in an active form. The enzyme seems to be an integral part of metaphase chromosomes and not merely a contamination of enzyme present in the cytoplasm [4], since metaphase chromosomes isolated by different methods yielded about the same result. Finally when isolated metaphase chromosomes were incubated in vitro with [14C]NAD, the chromosomes became ADP-ribosylated and hence could serve as an acceptor for ADP-ribosylation. This might suggest that ADPribosylation of metaphase chromosomes plays a role during mitosis.

A method for density gradient isoelectric focusing in small scale

Abstract A method for density gradient isoelectric focusing in small seale is described. It utilizes the same equipment as that ordinarily used for electrofocusing in polyacrylamide gels, thus allowing several samples to be analyzed in the same experiment and the simultaneous electrofocusing in density gradients and gels. Data concerning the stability of the pH gradient and the effect of salt and sample load on the focusing are given. Three different methods for fractionating the gradient are described; in one of these the frozen gradient is sliced with an ordinary gel alicer. This fractionation method is especially advantageous if the gradient contains zones of highly aggregated material, and it also allows the gradients to be stored frozen for later fractionation.

Method for complete separation of the high mobility group (HMG) proteins HMG I and HMG Y from HMG 14 and HMG 17 and a procedure for purification of HMG I and HMG Y

A purification procedure which separates the four low-molecular-weight high mobility group (HMG) proteins, HMG 14, 17, I and Y, is described. The procedure includes chromatography on phosphocellulose and Blue Sepharose combined with reversed-phase high-performance liquid chromatography. The blue Sepharose column separates HMG I and Y completely from HMG 14 and 17, and should therefore be an useful tool for the identification of these proteins which in several reports have been confused with HMG 14 and 17. HMG I and Y on the one hand and HMG 14 and 17 on the other exhibited considerable differences in their affinities for Blue Sepharose, probably reflecting fundamental differences in biological function.

Post-Translational Modification of the Low Molecular Weight HMG Chromosomal Proteins

The low molecular weight proteins HMG-14 and 17 (reference 1, and chapter by Goodwin et al., this volume) have been shown to undergo post-translational modifications such as phosphorylation, ADP-ribosylation, glycosylation, and acetylation. We will discuss each of these with particular emphasis on phosphorylation, which is the best studied post-translational modification of these proteins.