The S?/?M checkpoint at 37°C and the recovery of viability of the mutant pol δ ts3 require the crb2 + /rhp9 + gene in fission yeast

We have isolated a mutant in fission yeast, in which mitosis is uncoupled from completion of DNA replication when DNA synthesis is impaired by a thermosensitive mutation in the gene encoding the catalytic subunit of DNA polymerase δ. By functional complementation, we cloned the wild-type gene and identified it as the recently cloned checkpoint gene crb2 ⁺ /rhp9 ⁺ . This gene has been implicated in the DNA damage checkpoint and acts in the Chk1 pathway. Unlike the deleted strain dcrb2, cells bearing the crb2-1 allele were not affected in the DNA repair checkpoint after UV or MMS treatment at 30° C, but were defective in this checkpoint function when treated with MMS at 37° C. We analysed the involvement of Crb2 in the S/M checkpoint by blocking DNA replication with hydroxyurea, by using S phase cdc mutants, or by overexpression of the mutant PCNA L68S. Both crb2 mutants were unable to maintain the S/M checkpoint at 37° C. Furthermore, the crb2 ⁺ gene was required, together with the cds1 ⁺ gene, for the S/M checkpoint at 30° C. Finally, both the crb2 deletion and the crb2-1 allele induced a rapid death phenotype in the polδts3 background at both 30° C and 37° C. The rapid death phenotype was independent of the checkpoint functions. Received: 25 May 1998 / Accepted: 21 September 1998     

A novel yeast gene, RHK1 , is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits β-1,3-glucan synthesis

The HM-1 killer toxin from Hansenula mrakii is known to inhibit cell wall β-1,3-glucan synthase of Saccharomyces cerevisiae and other sensitive strains of yeast. A number of mutants of Saccharomyces cerevisiae that show resistance to this toxin were isolated in order to clarify the killing mechanism of the toxin. These mutants, designated rhk (resistant to Hansenula killer), were classified into three complementation groups. A novel gene RHK1, which complements the killer-resistant phenotype of the largest complementation group rhk1, was isolated. DNA sequence analysis revealed an open reading frame that encodes a hydrophobic protein composed of 458 amino acids. Gene disruption followed by tetrad analysis showed that RHK1 is not essential and loss of RHK1 function endowed S. cerevisiae cells with complete killer resistance. A biochemical analysis suggested that RHK1 does not participate directly in the synthesis of β-1,3-glucan but is involved in the synthesis of the receptor for the HM-1 killer toxin. Received: 27 June 1996 / Accepted: 14 October 1996     

The calcitonin/calcitonin gene related peptide-α gene is not required for 1α,25-dihydroxyvitamin D3-mediated suppression of experimental autoimmune encephalomyelitis

The active form of vitamin D, 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), can suppress disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Calcium appears to be a critical component of 1,25(OH)₂D₃-mediated suppression of EAE, as complete disease prevention only occurs with a concomitant increase in serum calcium levels. Calcitonin (CT) is a peptide hormone released in response to acute increases in serum calcium, which led us to explore its importance in 1,25(OH)₂D₃-mediated suppression of EAE. Previously, we discovered that co-administration of pharmacological doses of CT enhanced the suppressive effect of 1,25(OH)₂D₃ on EAE, suggesting CT may play a role in 1,25(OH)₂D₃-mediated suppression of EAE. To determine the importance of CT in EAE we have utilized a mouse strain in which the gene encoding CT and its alternative splice product, calcitonin gene related peptide-α (CGRP), have been deleted. Deletion of the CT/CGRP gene had no effect on EAE progression. Furthermore, treatment with 1,25(OH)₂D₃ suppressed EAE in CT/CGRP knock-out mice equal to that in wild type mice. Therefore, we conclude that CT is not necessary for 1,25(OH)₂D₃-mediated suppression of EAE.     

The IKKα-dependent NF-κB p52/RelB noncanonical pathway is essential to sustain a CXCL12 autocrine loop in cells migrating in response to HMGB1

HMGB1 is a chromatin architectural protein that is released by dead or damaged cells at sites of tissue injury. Extracellular HMGB1 functions as a proinflammatory cytokine and chemoattractant for immune effector and progenitor cells. Previously, we have shown that the inhibitor of NF-κB kinase (IKK)β- and IKKα-dependent NF-κB signaling pathways are simultaneously required for cell migration to HMGB1. The IKKβ-dependent canonical pathway is needed to maintain expression of receptor for advanced glycation end products, the ubiquitously expressed receptor for HMGB1, but the target of the IKKα non-canonical pathway was not known. In this study, we show that the IKKα-dependent p52/RelB noncanonical pathway is critical to sustain CXCL12/SDF1 production in order for cells to migrate toward HMGB1. Using both mouse bone marrow-derived macrophages and mouse embryo fibroblasts (MEFs), it was observed that neutralization of CXCL12 by a CXCL12 mAb completely eliminated chemotaxis to HMGB1. In addition, the HMGB1 migration defect of IKKα KO and p52 KO cells could be rescued by adding recombinant CXCL12 to cells. Moreover, p52 KO MEFs stably transduced with a GFP retroviral vector that enforces physiologic expression of CXCL12 also showed near normal migration toward HMGB1. Finally, both AMD3100, a specific antagonist of CXCL12's G protein-coupled receptor CXCR4, and an anti-CXCR4 Ab blocked HMGB1 chemotactic responses. These results indicate that HMGB1-CXCL12 interplay drives cell migration toward HMGB1 by engaging receptors of both chemoattractants. This novel requirement for a second receptor-ligand pair enhances our understanding of the molecular mechanisms regulating HMGB1-dependent cell recruitment to sites of tissue injury.     

The Saccharomyces cerevisiae protein YJR043C (Pol32) interacts with the catalytic subunit of DNA polymerase α and is required for cell cycle progression in G2?/?M

We have analysed the YJR043c gene of Saccharomyces cerevisiae, previously identified by systematic sequencing. The deletion mutant (yjr043cΔ) shows slow growth at low temperature (15° C), while at 30° C and 37° C the growth rate of mutant cells is only moderately affected. At permissive and nonpermissive temperatures, mutant cells were larger and showed a high proportion of large-budded cells with a single duplicated nucleus at or beyond the bud neck and a short spindle. This phenotype was even more striking at low temperature, the mutant cells becoming dumbbell shaped. All these phenotypes suggest a role for YJR043C in cell cycle progression in G2/M phase. In two-hybrid assays, the YJR043c gene product specifically interacted with Poll, the catalytic subunit of DNA polymerase α. The pol1-1 /yjr043cΔ double mutant showed a more severe growth defect than the pol1-1 single mutant at permissive temperature. Centromeric plasmid loss rate elevated in yjr043cΔ. Analysis of the sequence upstream of the YJR043c ORF revealed the presence of an MluI motif (ACGCGT), a sequence associated with many genes involved in DNA replication in budding yeast. The cell cycle phenotype of the yjr043cΔ mutant, the evidence for genetic interaction with Pol1, the presence of an MluI motif upstream and the elevated rate of CEN plasmid loss in mutants all support a function for YJR043C in DNA replication. Received: 22 July 1998 / Accepted: 22 September 1998