The Msd1–Wdr8–Pkl1 complex anchors microtubule minus ends to fission yeast spindle pole bodies

The minus ends of spindle microtubules are anchored to a microtubule-organizing center. The conserved Msd1/SSX2IP proteins are localized to the spindle pole body (SPB) and the centrosome in fission yeast and humans, respectively, and play a critical role in microtubule anchoring. In this paper, we show that fission yeast Msd1 forms a ternary complex with another conserved protein, Wdr8, and the minus end–directed Pkl1/kinesin-14. Individual deletion mutants displayed the identical spindle-protrusion phenotypes. Msd1 and Wdr8 were delivered by Pkl1 to mitotic SPBs, where Pkl1 was tethered through Msd1–Wdr8. The spindle-anchoring defect imposed by msd1/wdr8/pkl1 deletions was suppressed by a mutation of the plus end–directed Cut7/kinesin-5, which was shown to be mutual. Intriguingly, Pkl1 motor activity was not required for its anchoring role once targeted to the SPB. Therefore, spindle anchoring through Msd1–Wdr8–Pkl1 is crucial for balancing the Cut7/kinesin-5–mediated outward force at the SPB. Our analysis provides mechanistic insight into the spatiotemporal regulation of two opposing kinesins to ensure mitotic spindle bipolarity.     

Mapping of the interleukin 5 receptor gene to human Chromosome 3 p25–p26 and to mouse Chromosome 6 close to the Raf-1 locus with polymorphic tandem repeat sequences

Simple-sequence tandem repeat sequences in the 3 UTR of interleukin 5 (IL5)-receptor gene of human and mouse are polymorphic in their length among humans and different strains of mice. In 20 different human Epstein-Barr virus (EBV)-transformed cell lines, six alleles of IL5R could be distinguished. In the mouse, three different alleles are found. With the human-specific IL5R tandem repeat marker in human-rodent somatic cell hybrids, the IL5R gene was mapped to human Chromosome (Chr) 3 p25–p26. With the mouse-specific IL5R tandem repeat sequence in recombinant inbred strains of mice, the Il5r gene was mapped to the distal part of mouse Chr 6 close to the Raf-1 locus.     

The single DR β gene of the DRw8 haplotype is closely related to the DR β 3III gene encoding DRw52

In most individuals two HLA-DR genes are expressed from each chromosome. One of these genes encodes one of the classical DR specificities, while the other encodes either of the supertypic DRw52/DRw53 specificities. In addition to these genes usually one or two DR pseudogenes are present. In contrast, the DRw8 chromosomal region only contains a single DR gene. To determine the relationship of this single gene to the multiple DR genes of other DR specificities, comparisons of Southern genomic blots were carried out. In this analysis genomic clones for each individual DR chain locus were included. The DR w8 gene was indistinguishable from the DR III gene of DR3 cells (encoding DRw52), suggesting that it is closely related to the latter gene. The functional implications of this finding are discussed.     

Cloning and mapping of bovine ZFX gene to the long arm of the X-chromosome (Xq34) and homologous mapping of ZFY gene to the distal region of the short arm of the bovine (Yp13), ovine (Yp12–p13), and caprine (Yp12–p13) Y Chromosome

A part of mouse Zfy-2 sequence was synthesized and used to screen a genomic library of the spinous country-rat (Tokudaia osimensis spp., 2n = 45). An isolated clone had the C-terminal region of Zfy, which consisted of 1190 bp, encoded 336 amino acid residues, and harbored 11 out of 13 zinc finger motifs. With this as a probe, a bovine testis cDNA library was screened. Two ZFX clones were isolated and their sequences combined. The short sequence, lacking part of the 5′ upstream region, was amplified by PCR or RT-PCR, cloned, and sequenced. A full-length ZFX was constructed by combining these three sequences. The bovine ZFX consisted of 5328 bp and encoded 800 amino acid residues, which contained 13 zinc finger motifs. ZFX was used as a probe for fluorescence in situ hybridization and was mapped to Xq34, different from its previously reported site at Xq21-q231. A SINE (short interspersed nuclear element) sequence consisting of 188 bp was found close to the end of the 3′-untranslated region of ZFX. The SINE sequence hybridized to all bovine chromosomes. ZFY is highly homologous with ZFX and, as a result, ZFY could be mapped simultaneously. ZFY was mapped to the distal region of the short arm of the Y Chromosome (Chr) (Yp13), contradicting the previously reported position Yq1. Ovine and caprine ZFY were also mapped with bovine ZFX. Both were mapped to the distal region of the short arm of the Y Chr (Yp12-p13). Ovine ZFX was mapped to a region close to the centromere of the X Chr (Xq13). Received: 23 July 1997 / Accepted: 30 September 1997     

Structure–function analysis and genetic interactions of the Yhc1, SmD3, SmB,and Snp1 subunits of yeast U1 snRNP and genetic interactions of SmD3 with U2 snRNP subunit Lea1

Yhc1 and U1-C are essential subunits of the yeast and human U1 snRNP, respectively, that stabilize the duplex formed by U1 snRNA at the pre-mRNA 5′ splice site (5′SS). Mutational analysis of Yhc1, guided by the human U1 snRNP crystal structure, highlighted the importance of Val20 and Ser19 at the RNA interface. Though benign on its own, V20A was lethal in the absence of branchpoint-binding complex subunit Mud2 and caused a severe growth defect in the absence of U1 subunit Nam8. S19A caused a severe defect with mud2▵. Essential DEAD-box ATPase Prp28 was bypassed by mutations of Yhc1 Val20 and Ser19, consistent with destabilization of U1•5′SS interaction. We extended the genetic analysis to SmD3, which interacts with U1-C/Yhc1 in U1 snRNP, and to SmB, its neighbor in the Sm ring. Whereas mutations of the interface of SmD3, SmB, and U1-C/Yhc1 with U1-70K/Snp1, or deletion of the interacting Snp1 N-terminal peptide, had no growth effect, they elicited synthetic defects in the absence of U1 subunit Mud1. Mutagenesis of the RNA-binding triad of SmD3 (Ser-Asn-Arg) and SmB (His-Asn-Arg) provided insights to built-in redundancies of the Sm ring, whereby no individual side-chain was essential, but simultaneous mutations of Asn or Arg residues in SmD3 and SmB were lethal. Asn-to-Ala mutations SmB and SmD3 caused synthetic defects in the absence of Mud1 or Mud2. All three RNA site mutations of SmD3 were lethal in cells lacking the U2 snRNP subunit Lea1. Benign C-terminal truncations of SmD3 were dead in the absence of Mud2 or Lea1 and barely viable in the absence of Nam8 or Mud1. In contrast, SMD3-E35A uniquely suppressed the temperature-sensitivity of lea1▵.     

The fine-tuning of TRAF2–GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex

We provide the first biochemical evidence of a direct interaction between the glutathione transferase P1-1 (GSTP1-1) and the TRAF domain of TNF receptor-associated factor 2 (TRAF2), and describe how ligand binding modulates such an equilibrium. The dissociation constant of the heterocomplex is K_d=0.3 μM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (K_d≥2.6 μM) or is inactivated by oxidation (K_d=1.7 μM). This indicates that GSTP1-1''s TRAF2-binding region involves the GSH-binding site. The GSTP1-1 inhibitor NBDHEX further decreases the complex''s binding affinity, as compared with when GSH is the only ligand; this suggests that the hydrophobic portion of the GSTP1-1 active site also contributes to the interaction. We therefore hypothesize that TRAF2 binding inactivates GSTP1-1; however, analysis of the data, using a model taking into account the dimeric nature of GSTP1-1, suggests that GSTP1-1 engages only one subunit in the complex, whereas the second subunit maintains the catalytic activity or binds to other proteins. We also analyzed GSTP1-1''s association with TRAF2 at the cellular level. The TRAF2–GSTP1-1 complex was constitutively present in U-2OS cells, but strongly decreased in S, G2 and M phases. Thus the interaction appears regulated in a cell cycle-dependent manner. The variations in the levels of individual proteins seem too limited to explain the complex''s drastic decline observed in cells progressing from the G0/G1 to the S–G2–M phases. Moreover, GSH''s intracellular content was so high that it always saturated GSTP1-1. Interestingly, the addition of NBDHEX maintains the TRAF2–GSTP1-1 complex at low levels, thus causing a prolonged cell cycle arrest in the G2/M phase. Overall, these findings suggest that a reversible sequestration of TRAF2 into the complex may be crucial for cell cycle progression and that multiple factors are involved in the fine-tuning of this interaction.     

The gene coding for the p68 calcium-binding protein is localised to bands q32–q34 of human chromosome 5, and to mouse chromosome 11

Summary The gene encoding a tissue inhibitor of metalloproteinases, TIMP, has previously been shown to be X-linked in both the human and mouse genomes. We have used a series of somatic cell hybrids segregating translocation and deletion X chromosomes to map the TIMP gene on the human X chromosome. In combination with previous data, the gene can be assigned to Xp11.23Xp11.4. Genetic linkage analyses demonstrate that TIMP is linked to the more distal ornithine transcarbamylase (OTC) locus at a distance of about 22 centimorgans. The data are consistent with the conclusion that TIMP maps to a conserved synteny and linkage group on the proximal short arm of the human X chromosome and on the pericentric region of the mouse X chromosome, including loci for synapsin-1, a member of the raf oncogene family, OTC, and TIMP.