Minimal ectopic expression of a 58-kDa protein kinase (PITSLRE beta 1), distantly related to members of the cdc2 gene family, induces telophase delay, abnormal chromosome segregation, and decreased growth rates in Chinese hamster ovary cells. Here we show that this decrease in cell growth rate is due to apoptosis. Apoptosis is also induced by ectopic expression of an amino-terminal deletion mutant containing the catalytic and C-terminal domains of PITSLRE beta 1 but not by other mutants lacking histone H1 kinase activity or by other members of the cdc2 gene family. However, unlike the wild-type PITSLRE beta 1 over-expressors, ectopic expression of the N-terminal PITSLRE beta 1 mutant does not result in telophase delay or abnormal chromosome segregation. These results suggested that the function of this protein kinase could be linked to apoptotic signaling. To test this hypothesis, we examined levels of PITSLRE mRNA, steady-state protein, and enzyme activity in human T cells undergoing apoptosis after activation with the anti-Fas monoclonal antibody (MAb). All were substantially elevated shortly after Fas MAb treatment. In addition to new transcription and translation, proteolysis contributed to the increased steady-state levels of a novel 50-kDa PITSLRE protein, as suggested by the diminution of larger PITSLRE isoforms observed in the same cells. Indeed, treatment of the Fas-activated T cells with a serine protease inhibitor prevented apoptotic death and led to the accumulation of larger, less active PITSLRE kinase isoforms but not the enzymatically active 50-kDa PITSLRE isoform. Finally, induction of apoptosis by glucocorticoids in the same cell line, as well as by Fas MAb treatment of another T-cell line, led to a similar induction of 50-kDa PITSLRE protein levels over time. These findings suggest that (i) PITSLRE kinase(s) may lie within apoptotic signaling pathway(s), (ii) serine protease activation may be an early event in Fas-activated apoptosis of human T cells, and (iii) some PITSLRE kinase isoforms may be targets of apoptotic proteases.

We have isolated and characterized cDNA encoding a human 58-kDa protein kinase that is homologous to the cell division control (CDC) protein kinases. This protein kinase also contains a unique N-terminal domain that may potentially regulate its function. Due to its relatedness to p34CDC2, the human p58 cDNA was overexpressed in CHO cells to determine the effect on the cell cycle. Elevated expression of p58 in these cells resulted in prolonged late telophase and early G1 phase of the cell cycle. These p58 overexpressors showed a significantly increased frequency of tubulin midbodies as well as significant increases in mitotic abnormalities. Thus, proper regulation ofp58 protein kinase is essential for normal cell cycle progression in these cells.

In Escherichia coli K-12, the pyrBI operon encodes the two nonidentical subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase (ATCase). Expression of the pyrBI operon is regulated over an -300-fold range by pyrimidine availability. Most of this regulation (i.e., 50-fold) occurs through a UTP-sensitive attenuation control mechanism in which low intracellular levels of UTP cause transcriptional pausing within a segment of the pyrBI leader region that specifies a 44-codon open reading frame. This pausing permits close coupling of transcription and translation within the leader region, which suppresses transcriptional termination at an attenuator (a p-independent transcriptional terminator) located,in front of the pyrB structural gene (Turnbough et al. 1983;Roland et al. 1988;Liu and Turnbough 1989;Landick and Turnbough 1992; Donahue and Turnbough 1994).Pyrimidine-mediated regulation of pyrBI expression also occurs at the level of transcriptional initiation (Don1Corresponding author. ahue and Turnbough 1990;Liu et al. 1993). Recent studies have suggested that this regulation involves a run of three T-A base pairs (specifying UUU) in the initially transcribed region of the pyrBI promoter (Donahue and Turnbough 1990). The pyrBI promoter region contains the sequence 5'-TATAATGCCGGACAATTTGCCG; the -10 region and two adenine residues at which transcriptional initiation can occur in vitro are underlined, and the more downstream adenine residue appears to be the predominant transcriptional start site in vivo (Donahue and Turnbough 1990). It was discovered that RNA polymerase formed heparin-resistant, transcriptionally competent initiation complexes at the pyrBI promoter in the presence of ATP and CTP and, to a small extent, with ATP alone, indicating stabilization by the nascent transcripts ACAA and AA, respectively. In contrast, in the presence of ATP and UTP, RNA polymerase did not form initiation complexes that were capable of synthesizing pyrBI transcripts following a heparin challenge.This result suggested that the synthesis of a nascent transcript with the sequence AAUUU did not stabilize the initiation complex or perhaps interfered with pro-

Staphylococcus simulans biovar staphylolyticus produces an extracellular glycylglycine endopeptidase (lysostaphin) that lyses other staphylococci by hydrolyzing the cross bridges in their cell wall peptidoglycans. The genes for endopeptidase (end) and endopeptidase resistance (epr) reside on plasmid pACK1. An 8.4-kb fragment containing end was cloned into shuttle vector pLI50 and was then introduced into Staphylococcus aureus RN4220. The recombinant S. aureus cells produced endopeptidase and were resistant to lysis by the enzyme, which indicated that the cloned fragment also contained epr. Treatments to remove accessory wall polymers (proteins, teichoic acids, and lipoteichoic acids) did not change the endopeptidase sensitivity of walls from strains of S. simulans biovar staphylolyticus or of S. aureus with and without epr. Immunological analyses of various wall fractions showed that there were epitopes associated with endopeptidase resistance and that these epitopes were found only on the peptidoglycans of epr ؉ strains of both species. Treatment of purified peptidoglycans with endopeptidase confirmed that resistance or susceptibility of both species was a property of the peptidoglycan itself. A comparison of the chemical compositions of these peptidoglycans revealed that cross bridges in the epr ؉ cells contained more serine and fewer glycine residues than those of cells without epr. The presence of the 8.4-kb fragment from pACK1 also increased the susceptibility of both species to methicillin.

No abstract