Sex ratio and intrasexual kin competition in mammals

Summary Asymmetries in both intrasexual competition and generation overlap occur in Antechinus (Dasyuridae; Marsupialia). We show that the range of interpopulation variation in the sex ratio of pouch young spans and exceeds the range of sex ratios at birth hitherto recorded from eutherians (Clutton-Brock and Albon 1983). Although postweaning dispersal and male mortality are similar among all Antechinus populations, interpopulation variance in female longevity leads to variable duration of mother/daughter interaction. As this duration increases, parental investment is increasingly biased toward males, supporting the view that local competition among female kin for resources may influence mammalian sex ratios.     

Ketone-body metabolism after partial hepatectomy in the rat.

Fed or 24 h-starved rats were subjected to two-thirds partial hepatectomy or sham-operation and subsequently starved for 4, 14 or 24 h. Despite high plasma fatty acid concentrations, the partially hepatectomized rats failed to respond to post-operative starvation with increased blood and liver ketone-body concentrations or to maintain the high ketone-body concentrations associated with pre-operative starvation. Hypoglycaemia and hyperlactaemia were observed within 30 min of functional hepatectomy, but not partial hepatectomy, of 24 h-starved rats, and, even after a further 24 h starvation of partially hepatectomized rats, blood glucose concentrations were only slightly decreased. The results are discussed with reference to fat oxidation and gluconeogenesis in the liver remaining after partial hepatectomy.     

Isolation of an actin polymerization stimulator from bovine thyroid plasma membranes

An actin polymerization stimulator was purified from bovine thyroid plasma membranes by DNase I affinity column chromatography. Although the molecular weight of the protein was about 42,000 (42K) by sodium dodecyl sulfate polyacrylamide gel electrophoresis, it did not comigrate with actin. In the presence of 30 mM KCl, the 42K protein facilitated formation of actin filaments when analyzed by a centrifugation method, accelerated the initial phase of actin polymerization as measured in an Ostwald viscometer and increased the length of filaments as shown by electron microscopy. The 42K protein also accelerated the initial phase of actin polymerization in the presence of 100 mM KCl and 2 mM MgCl₂ but did not affect the final viscosity. The effect of the 42K protein was diminished by 5 uM cytochalasin B or 1 uM cytochalasin D. This 42K protein may anchor actin filaments onto the thyroid plasma membrane.     

Definition of subchromosomal intervals around the myotonic dystrophy gene region at 19q

The localization to 19q of the gene causing myotonic dystrophy (DM) has been defined more precisely by refinement of the physical location of several linked markers. A somatic cell hybrid mapping panel from cells with t(1;19), t(12;19), and t(X;19) translocation products was constructed to define five different intervals across 19q. In addition, we have derived a series of cell hybrids by irradiation of a der(19)-only hybrid to further subdivide the cen-q13.1 region. Using an array of 36 cloned genes, anonymous DNAs, and enzyme markers, we have tested the location of the panel breakpoints and refined the regional assignment of several of these markers. All markers tightly linked to DM are localized mainly within 19q13.2, thus suggesting that the DM gene is also close to this region.     

A Role for the Disintegrin Domain of Cyritestin, a Sperm Surface Protein Belonging to the ADAM Family, in Mouse Sperm–Egg Plasma Membrane Adhesion and Fusion

Sperm–egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell–cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm–egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (α and β subunits). Fertilin α and β are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin β functions in sperm–egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin α, fertilin β, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin β, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm–egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80–90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin β active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin β also strongly inhibited (80–90%) both sperm–egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin β showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin β in sperm–egg plasma membrane adhesion leading to fusion.     

Isolation and characterization of functional domains of UvrA.

The sequence of Escherichia coli UvrA protein suggests that it may fold into two functional domains each possessing DNA binding and ATPase activities. We have taken two approaches to physically isolate polypeptides corresponding to the two putative domains. First, a 180 base pair DNA segment encoding multiple collagenase recognition sequences was inserted into UvrA's putative interdomain hinge region. This UvrA derivative was purified and digested with collagenase, and the resulting 70-kDa N-terminal and 35-kDa C-terminal fragments were purified. Both fragments possessed nonspecific DNA binding activity, but only the N-terminal domain retained its nucleotide binding capacity as evidence by measurements of ATP hydrolysis and by ATP photo-cross-linking. Together, the two fragments failed to substitute for UvrA in reconstituting (A)BC excinuclease and, therefore, were presumed to be unable to load UvrB onto damaged DNA. Second, the DNA segments encoding the two domains were fused to the beta-galactosidase gene. The UvrA N-terminal domain-beta-galactosidase fusion protein was overproduced and purified. This fusion protein had ATPase activity, thus confirming that the amino-terminal domain does possess an intrinsic ATPase activity independent of any interaction with the carboxy terminus. Our results show that UvrA has two functional domains and that the specificity for binding to damaged DNA is provided by the proper three-dimensional orientation of one zinc finger motif relative to the other and is not an intrinsic property of an individual zinc finger domain.     

Site-specific mutagenesis of conserved residues within Walker A and B sequences of Escherichia coli UvrA protein.

UvrA is the ATPase subunit of the DNA repair enzyme (A)BC excinuclease. The amino acid sequence of this protein has revealed, in addition to two zinc fingers, three pairs of nucleotide binding motifs each consisting of a Walker A and B sequence. We have conducted site-specific mutagenesis, ATPase kinetic analyses, and nucleotide binding equilibrium measurements to correlate these sequence motifs with activity. Replacement of the invariant Lys by Ala in the putative A sequences indicated that K37 and K646 but not K353 are involved in ATP hydrolysis. In contrast, substitution of the invariant Asp by Asn in the B sequences at positions D238, D513, or D857 had little effect on the in vivo activity of the protein. Nucleotide binding studies revealed a stoichiometry of 0.5 ADP/UvrA monomer while kinetic measurements on wild-type and mutant proteins showed that the active form of UvrA is a dimer with 2 catalytic sites which interact in a positive cooperative manner in the presence of ADP; mutagenesis of K37 but not of K646 attenuated this cooperativity. Loss of ATPase activity was about 75% in the K37A, 86% in the K646A mutant, and 95% in the K37A-K646A double mutant. These amino acid substitutions had only a marginal effect on the specific binding of UvrA to damaged DNA but drastically reduced its ability to deliver UvrB to the damage site. We find that the deficient UvrB loading activity of these mutant UvrA proteins results from their inability to associate with UvrB in the form of (UvrA)2(UvrB)1 complexes. We conclude that UvrA forms a dimer with two ATPase domains involving K37 and K646 and that the work performed by ATP hydrolysis is the delivery of UvrB to the damage site on DNA.     

Structural organization of the genome of Dictyostelium discoideum: Analysis by EcoR1 restriction endonuclease

The genome of the cellular slime mold Dictyostelium discoideum has been analyzed by limit digestion with EcoR1 restriction endonuclease. Approximately 15% of the nuclear genome is cleaved into nine discrete fragments as analyzed by agarose gel electrophoresis. These fragments appear to be derived from two nuclear buoyant density satellites, one of which contains sequences coding for ribosomal RNA. The bulk of the nuclear DNA is digested into approximately 7000 fragments with a mean molecular weight of 4 × 10⁶ to 5 × 10⁶. The mitochondrial DNA is digested into four fragments. One of the nuclear bands has been cloned in Escherichia coli using plasmid pSC101 carrying tetracyline resistance. Analysis by renaturation kinetics indicates that it is repeated approximately 200 times per haploid genome and that it is not internally repeated.     

α-Mannosidase-2: A developmentally regulated enzyme in Dictyostelium discoideum

A previously undected isozyme of α-mannosidase was observed in several independent mutant strains of Dictyostelium discoideum selected for the absence of the major isozyme, α-mannosidase-1. The activity in the mutant strains, α-mannosidase-2, differs from the major isozyme with respect to pH optimum, substrate affinity, sensitivity to inhibition by l-cysteine, and is particulate bound. The enzyme can be solubilized by treatment of the extract with nonionic detergents. α-Mannosidase-2 begins to accumulate only after 12 hr of development and reaches a peak specific activity of about a tenth of that of α-mannosidase-1 during culmination. The increase in specific activity of α-mannosidase-2 is blocked by either cycloheximide or actinomycin D, drugs known to inhibit protein and RNA synthesis, respectively, and probably results from accumulation of de novo synthesized enzyme. α-Mannosidase-2, therefore, provides a convenient marker enzyme for biochemical differentiation during the pseudo-plasmodial stage.