Sap1, a protein that binds to sequences required for mating-type switching, is essential for viability in Schizosaccharomyces pombe.

The pattern of mating-type switching in cell pedigrees of the fission yeast Schizosaccharomyces pombe is dictated by the inheritance of specific DNA chains at the mating-type locus (mat1). The recombination event essential for switching is initiated by a site-specific double-strand break at mat1. The switch-activating protein, Sap1, binds in vitro to a mat1 cis-acting site that was shown earlier to be essential for efficient mating-type switching. We isolated the sap1 gene by using oligonucleotides corresponding to the amino acid sequence of purified Sap1 protein. The sequence of that gene predicted a 30-kDa protein with no significant homology to other canonical DNA-binding protein motifs. To facilitate its biochemical characterization, Sap1 was expressed in Escherichia coli. The protein expressed in bacteria displayed the same DNA-binding specificities as the protein purified from S. pombe. Interestingly, analysis of a sap1 null mutation showed that the gene is essential for growth even in a strain in which mating-type switching is prohibited because of a defect in generation of the double-strand break. Thus, the sap1 gene product implicated in mating-type switching is shown to be essential for cell viability.     

Directionality of Fission Yeast Mating-Type Interconversion Is Controlled by the Location of the Donor Loci

Cells of homothallic strains of Schizosaccharomyces pombe efficiently switch between two mating types called P and M. The phenotypic switches are due to conversion of the expressed mating-type locus (mat1) by two closely linked silent loci, mat2-P and mat3-M, that contain unexpressed information for the P and M mating types, respectively. In this process, switching-competent cells switch to the opposite mating type in 72-90% of the cell divisions. Hence, mat2-P is a preferred donor of information to mat1 in M cells, whereas mat3-M is a preferred donor in P cells. We investigated the reason for the donor preference by constructing a strain in which the genetic contents of the donor loci were swapped. We found that switching to the opposite mating type was very inefficient in that strain. This shows that the location of the silent cassettes in the chromosome, rather than their content, is the deciding factor for recognition of the donor for each cell type. We propose a model in which switching is achieved by regulating accessibility of the donor loci, perhaps by changing the chromatin structure in the mating-type region, thus promoting an intrachromosomal folding of mat2 or mat3 onto mat1 in a cell type-specific fashion. We also present evidence for the involvement of the Swi6 and Swi6-mod trans-acting factors in the donor-choice mechanism. We suggest that these factors participate in forming the proposed folded structure.     

The Action of Homothallism Genes in Saccharomyces Diploids during Vegetative Growth and the Equivalence of hma and HMalpha Loci Functions

The action of homothallism genes in vegetatively growing diploid cells was examined. The results demonstrate that homothallism genes function during regular vegetative growth cycles as well as during the first few divisions after spore germination. A procedure based on ultraviolet-induced reciprocal mitotic recombination monitored by homozygosity for cryptopleurine resistance (a recessive marker closely linked to the mating-type locus) allowed us to identify and recover Saccharomyces cerevisiae colonies sectored for the mating-type locus i.e., a/a and alpha/alpha. Homothallism genes can switch a/a or alpha/alpha vegetative diploid cells, generated from a strain with genotype a/alpha HO/ho HMalpha/HMalpha HMa/HMa, to a/alpha diploids or a/a/alpha/alpha tetraploids during a given mitotic division cycle. We found that both a/a and alpha/alpha sectors generated from a strain with genotype a/alpha HO/HO hmalpha/hmalpha hma/HMa switch to a/alpha diploids or a/a/alpha/alpha tetraploids. This finding supports Naumov and Tolstorukov's suggestion (1973) that the hm a allele provides for the same functions as the HMalpha allele, namely, a switch at the mating-type locus from alpha to a. The HO allele is dominant to ho but hma and HMa alleles are codominant. A loose linkage between the mating-type and the HMalpha loci ( approximately 55cM), confirming Harashima, Nogi and Oshima (1974) data, was observed.     

Familial fragility on chromosome 16 (Fra 16q22) enhanced by both interferon and distamycin A

A family with a "fragile site" at 16q22, inducible by both interferon and Distamycin A, is reported. Immunological problems were found in the family. In a sibship of ten, eight children had died in infancy. Our study led to the conclusions that interferon and Distamycin A induce fragility at the same site, which has the same characteristics as the spontaneous fragile site; that a viral hypothesis for this fragility may be supported; and that immunoincompetence of one kind or another must be considered in families presenting a fragile site at 16q22.     

Precise Mapping of the Homothallism Genes HML and HMR in SACCHAROMYCES CEREVISIAE

The HML and HMR loci carry unexpressed copies of MATa and MATα information, and a replica of that information is transposed to MAT during mating-type interchange in Saccharomyces yeasts. A negative control mechanism keeps silent the information located at the HML and HMR loci. We mapped these loci by constructing strains in which these loci are expressed. In these strains, the mating type of the segregants is dependent upon the allele at HML and HMR. This novel approach is independent of their switching function. HML is located on the left arm of chromosome III distal to his4 by about 26.8 centimorgans (cM). HMR maps on the right arm of the same chromosome distal to thr4 by about 39.8 cM and proximal to MAL2 by about 1.0 cM. The results allow the exact placement of these loci and are in accord with the observations made by Harashima and Oshima (1976).     

MAR1-a Regulator of the HMa and HMalpha Loci in SACCHAROMYCES CEREVISIAE

A mutation in the MAR1 (mating-type regulator) locus causing sterility in Saccharomyces cerevisiae is reported. The mutation maps on the left arm of linkage group IV between trp1 and cdc2 at a distance of about 27 cM from trp1 and about 31 cM from cdc2. Haploid strains with genotype MATalpha HMalpha HMa mar1-1 and MATa HMalpha HMamar1-1 are sterile. However, MATalpha hmalpha HMa mar1-1 and MATa HMalpha hma mar1-1 strains exhibit alpha and a mating type, respectively. The sterile strains can be "rare mated" with standard strains as a consequence of mutational changes at HMa and HMalpha. It is proposed that the MAR1 locus blocks the expression of MATalpha and MATa information thought to exist at HMa and HMalpha loci, respectively (Hicks, Strathern and Herskowitz, 1977). In a mar1-1 mutant, the expression of both HMalpha and HMa information leads to a nonmating phenotype similar to that of MATa/MATalpha diploids. The genetic evidence reported here is consistent with a central feature of the "cassette model", namely that HMalpha and hma carry MATa information and HMa and hmalpha carry MATalpha information.     

Calcium ions and osteoclastogenesis initiate the induction of bone formation by coral‐derived macroporous constructs

Coral-derived calcium carbonate/hydroxyapatite macroporous constructs of the genus Goniopora with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) initiate the induction of bone formation. Which are the molecular signals that initiate pattern formation and the induction of bone formation? To evaluate the role of released calcium ions and osteoclastogenesis, 7% HA/CC was pre-loaded with either 500 μg of the calcium channel blocker, verapamil hydrochloride, or 240 μg of the osteoclast inhibitor, biphosphonate zoledronate, and implanted in the rectus abdominis muscle of six adult Chacma baboons Papio ursinus. Generated tissues on days 15, 60 and 90 were analysed by histomorphometry and qRT-PCR. On day 15, up-regulation of type IV collagen characterized all the implanted constructs correlating with vascular invasion. Zoledronate-treated specimens showed an important delay in tissue patterning and morphogenesis with limited bone formation. Osteoclastic inhibition yielded minimal, if any, bone formation by induction. 7% HA/CC pre-loaded with the Ca⁺⁺ channel blocker verapamil hydrochloride strongly inhibited the induction of bone formation. Down-regulation of bone morphogenetic protein-2 (BMP-2) together with up-regulation of Noggin genes correlated with limited bone formation in 7% HA/CC pre-loaded with either verapamil or zoledronate, indicating that the induction of bone formation by coral-derived macroporous constructs is via the BMPs pathway. The spontaneous induction of bone formation is initiated by a local peak of Ca⁺⁺ activating stem cell differentiation and the induction of bone formation.