The Effect of Novel Chromosome Position and Variable Dose on the Genetic Behavior of the Responder (Rsp) Element of the Segregation Distorter (Sd) System of Drosophila Melanogaster

In the Segregation distorter (SD) system of meiotic drive, a minimum of two trans-acting elements [Sd and E(SD)] act in concert to cause a certain probability of dysfunction for sperm carrying a sensitive allele at the Responder (Rsp) target locus. By employing a number of insertional translocations of autosomal material into the long arm of the Y chromosome, Rsp can be mapped as the most proximal locus in the 2R heterochromatin as defined both by cytology and lethal complementation tests. Several of these insertional translocations result in the transposition of Rsp to the Y chromosome, where its sensitivity remains virtually unaltered. This argues that Rsp is separable from the second chromosome centromere, that its behavior does not depend on its gross chromosomal position, and that meiotic pairing of the chromosomes carrying the various SD elements is not a prerequisite for sperm dysfunction. Several other translocations apparently leave both resulting chromosomes at least partially sensitive to SD action, suggesting that Rsp is a large subdivisible genetic element. This view is compatible with observations published elsewhere that suggest that Rsp is a cytologically large region of highly repetitive AT-rich DNA. The availability of Y-linked copies of Rsp also allows the construction of SD males carrying two independently segregating Rsp alleles; this in turn allows the production of sperm with zero, one or two Rsp copies from the same male. Examination of the relative recovery proportions of progeny arising from these gametes suggests that sperm with two Rsp copies survive at much lower frequencies than would be predicted if each Rsp acted independently in causing sperm dysfunction. Possible explanations for such behavior are discussed.     

Transposition of the Responder Element (Rsp) of the Segregation Distorter System (Sd) to the X Chromosome in Drosophila Melanogaster

In order to test whether the meiotic drive system Segregation distorter (SD) can operate on the X chromosome to exclude it from functional sperm, we have transposed the Responder locus (Rsp) to this element. This was accomplished by inducing detachments of a compound-X chromosome in females carrying a Y chromosome bearing a Rsps allele. Six Responder-sensitive-bearing X chromosomes, with kappa values ranging from 0.90 to 1.00, were established as permanent lines. Two of these have been characterized more extensively with respect to various parameters affecting meiotic drive. SD males with a Responder-sensitive X chromosome produce almost exclusively male embryos, while those with a Rsp-Y chromosome produce almost exclusively female embryos. This provides a genetic system of great potential utility for the study of early sex-specific differentiation events as it allows the collection of large numbers of embryos of a given sex.     

Antagonism of estrogen- and antiestrogen-induced uterine complement component C3 expression by ICI 164,384

The uterus of the immature rat synthesizes and secretes complement component C3 in response to estradiol treatment. This response occurs in the uterine epithelial cells and is also stimulated by several antiestrogens including tamoxifen and LY117018. The administration of a new antiestrogen ICI 164,384 blocked the estradiol as well as the antiestrogen-stimulated increases in uterine weight, epithelial cell height, C3 synthesis and C3 mRNA. ICI 164,384 demonstrated no agonist properties in terms of epithelial cell response as determined by C3 expression.     

Monoclonal antibody to rat uterine peroxidase and its use in identification of the peroxidase as being of eosinophil origin

Peroxidase was purified from uteri of estrogen-treated rats by calcium chloride extraction, affinity chromatography on concanavalin A-Sepharose and hydrophobic interaction chromatography on phenyl-Sepharose. An overall purification of greater than 1700-fold was achieved with a final recovery of 27%. Monoclonal antibodies to peroxidase were subsequently prepared by immunization of male C57BL/10J mice with the highly purified peroxidase from rat uterus. Spleen and lymph node cells from the mice were fused with Sp2/0-Ag 14 mouse myeloma cells. The resultant hybrid cells were screened for production of antibody using a solid-phase, double antibody radioimmunoassay. The mature rat spleen, shown previously to be abundant in eosinophils, contains high peroxidase activity. Spleen peroxidase purified by the same procedure as the uterine enzyme cross-reacted with a monoclonal antibody, designated IgG-107B, used in all subsequent studies. Peroxidase extracted from isolated rat eosinophils also cross-reacted with the antibody and yielded identical titers as the spleen and uterine peroxidases. Spleen, uterine and horse eosinophil peroxidase had the same apparent molecular weight, 57000, as determined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. Following electrophoretic transfer to nitrocellulose, spleen, uterine and eosinophil peroxidase reacted with monoclonal antibody, using an immunoblotting technique. These results provide biochemical and immunological evidence that the majority of the calcium chloride-extractable peroxidase activity from the uteri of estrogen-treated rats is derived from infiltrating eosinophils.     

Experimental Population Genetics of Meiotic Drive Systems I. Pseudo-Y Chromosomal Drive as a Means of Eliminating Cage Populations of DROSOPHILA MELANOGASTER

The experimental population genetics of Y-chromosome drive in Drosophila melanogaster is approximated by studying the behavior of T(Y;2),SD lines. These exhibit "pseudo-Y" drive through the effective coupling of the Y chromosome to the second chromosome meiotic drive locus, Segregation distorter (SD). T(Y;2),SD males consequently produce only male offspring. When such lines are allowed to compete against structurally normal SD⁺ flies in population cages, T(Y;2),SD males increase in frequency according to the dynamics of a simple haploid selection model until the cage population is eliminated as a result of a deficiency in the number of adult females. Cage population extinction generally occurs within about seven generations.—Several conclusions can be drawn from these competition cage studies:

(1) Fitness estimates for the T(Y;2),SD lines (relative to SD⁺ ) are generally in the range of 2–4, and these values are corroborated by independent estimates derived from studies of migration-selection equilibrium.

(2) Fitness estimates are unaffected by cage replication, sample time, or the starting frequency of T(Y;2),SD males, indicating that data from diverse cages can be legitimately pooled to give an overall fitness estimate.

(3) Partitioning of the T(Y;2),SD fitnesses into components of viability, fertility, and frequency of alternate segregation (Y + SD from X + SD⁺) suggests that most of the T(Y;2),SD advantage derives from the latter two components. Improvements in the system might involve increasing both the viability and the alternate segregation to increase the total fitness.

While pseudo-Y drive operates quite effectively against laboratory stocks, it is less successful in eliminating wild-type populations which are already segregating for suppressors of SD action. This observation suggests that further studies into the origin and rate of accumulation of suppressors of meiotic drive are needed before an overall assessment can be made of the potential of Y-chromosome drive as a tool for population control.

     

Responder (Rsp) alleles in the segregation distorter (SD) system of meiotic drive in Drosophila may represent a complex family of satellite repeat sequences

In D. melanogaster males carrying Segregation Distorter (SD) second chromosomes, sperm receiving sensitive alleles of the Responder (Rsp) locus are subject to high rates of dysfunction. The Rsp region is located in 2R immediately adjacent to the centromere in heterochromatic band 39, and covers roughly 600 kb of material, of which approximately 85 kb is comprised of several hundred copies of a 240-bp satellite DNA sequence. Cytological observations as well as molecular analysis of rearrangements which bisect h39 indicate that sensitivity of the Rsp target to SD action is also subdivisible, and sensitivities of the component pieces appear to be correlated with copy number of the 240 bp repeat. In an attempt to examine possible higher order sequence structure for these blocks, PCR using single primers derived from a canonical repeat was used to identify potential reversals of direction of tandem arrays; that is, head-to-head or tail-to-tail junctions. Surprisingly, for two different Rsp alleles, only a single such reversal product for each was identified, differing in size and sequence between alleles. Sequencing of PCR products identified diverged copies of the canonical repeats that would not have been found using the levels of DNA stringency employed in earlier studies. Examination of Southern digests and slot-blots for DNA quantification indicates that adding the estimated numbers of such diverged copies to the canonical repeat copies discovered earlier is potentially sufficient to account for the entire 600 kb Rsp region. This adds strength to the hypothesis that this extended family of repeats is in fact the target of SD-mediated sperm dysfunction. Implications of these results for understanding the evolution of repetitive DNA are also discussed.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

Leukocyte chemotactic activity of cyclophilin.

During the purification of eosinophil chemotactic factors synthesized by the uterus in response to estrogen we isolated a protein having an N-terminal (15 amino acids) sequence identical to that of rat cyclophilin. Our data demonstrate that cyclophilin, a cytosolic protein isolated from bovine thymocytes, which specifically binds the immunosuppressive drug cyclosporin A, as well as recombinant human cyclophilin, displays eosinophil chemotactic activity. In addition to its chemotactic activity, cyclophilin stimulated the release of peroxidase activity from eosinophils. Maximal chemotactic activity of cyclophilin was achieved at a concentration of approximately 10 nM. At similar concentrations cyclophilin was also able to stimulate the migration of neutrophils. This chemotactic activity could be prevented by the addition of cyclosporin A, but not by a nonimmunosuppressive analog (1-fur-furyl-cyclosporin A) at similar concentrations. This chemotactic activity may represent an additional mechanism by which immunosuppressive drugs function to prevent tissue rejection.