Biogenesis of podocalyxin--the major glomerular sialoglycoprotein--in the newborn rat kidney

The appearance and distribution of podocalyxin on the glomerular epithelium (podocytes) during glomerular development was determined in the newborn rat kidney using specific monoclonal and affinity-purified polyclonal antibodies. Kidneys from 2-day-old rats were perfusion-fixed and processed for immunofluorescence or immunoperoxidase localization or immunogold labeling on ultrathin frozen sections. Podocalyxin first appeared on the apical surfaces of the presumptive podocytes of the S-shaped body above the level of the junctional complexes that connect the cells at this stage. The latter consist of a shallow occluding zonule and a deeper adhering zonule. Early in the capillary loop stage, when the urinary spaces open and the junctional complexes migrate from the apex to the base of the cells, labeling for podocalyxin extended along the lateral plasmalemma above the migrating junctions. In the maturing glomerulus when the foot processes form and the occluding and adhering junctions give way to developing slit diaphragms, podocalyxin was found along all newly-opened surfaces above the occluding junctions or slit membranes. No labeling was found below the latter. Podocalyxin was also detected intracellularly throughout the entire exocytotic pathway--i.e., in the rough endoplasmic reticulum and perinuclear cisternae, in Golgi cisternae and associated vesicles, and in carrier vesicles presumably en route to the cell surface. It is concluded that 1) podocalyxin is synthesized at a high rate in the differentiating podocyte; 2) its distribution is restricted to the apical plus lateral plasmalemmal domain facing the urinary spaces above the migrating junctions; 3) its time of appearance and distribution during glomerular development are identical to that reported earlier for epithelial polyanion; and 4) its synthesis and insertion into the podocyte plasmalemma is closely coupled to the development of the foot processes and filtration slits.     

Synthesis and In vitro Biological Activity of Cyclic Lipophilic χ-Conotoxin MrIA Analogues

The 13-residue peptide, χ-conotoxin MrIA extracted from the venom of Conus marmoreus, is a potent and selective inhibitor of the human noradrenaline transporter (NET). With the aim of improving its biophysical properties, chemical modifications were performed including the attachment of a lipophilic amino acid at the N-terminus and cyclisation of the peptide backbone with functionality introduced into the linker. All χ-conotoxin MrIA analogues were assembled on solid phase by highly optimised Boc chemistry and N- to C-cyclic analogues accessed by cysteine-mediated intramolecular native chemical ligation. In vitro biological activity at the human NET was evaluated by functional assays. All analogues inhibited the uptake of [³H]noradrenaline with comparable potencies to that of the native peptide, with one of the analogues, the linear N-terminal aminotetradecanoyl MrIA showing a 3-fold increase in potency (p < 0.05).     

Chromosome dynamics in rad12 mutants of Coprinus cinereus

We have characterized the phenotypes of three rad12 mutants of the basidiomycete Coprinus cinereus, which were isolated on the basis of sensitivity to ionizing radiation. Electron microscopic studies of meiotic nuclear spreads showed that all three rad12 mutants are defective in chromosomal synapsis. For rad12-1 and rad12-4, very limited assembly of the synaptonemal complex occurs. The phenotype of rad12-15 is less severe and longer stretches of synapsed chromosomes are formed. However, for all three alleles mutant nuclei arrest in a diffuse state with little synaptonemal complex structure. Observations made of spreads of acridine orange-stained meiotic nuclei correlated with the electron microscopic data. In rad12 strains, chromosomes condense but do not pair, and they later arrest in a decondensed state; very few rad12 cells enter metaphase I. Homozygous dikaryons of rad12 mutants produce fruiting bodies with significantly fewer basidiospores than are found in wild-type dikaryons. The viability of these spores is greatly reduced: all spores produced by rad12-1 and rad12-4 mushrooms fail to germinate, while only 16% of rad12-15 spores are viable. Recombination within the tract of the ribosomal RNA gene repeats was not significantly different in the mutants when compared with a wild-type congenic control. Quantitative measurements of oidial survival indicate that all three rad12 alleles are sensitive to gamma radiation but insensitive to UV radiation relative to wild-type strains.     

Homolog pairing and meiotic progression in Coprinus cinereus

We have used fluorescence in situ hybridization to examine homolog pairing during the synchronous meiosis of the basidiomycete Coprinus cinereus. Using spread preparations of meiotic nuclei, we confirmed previous studies that showed that at 6 h post-karyogamy essentially all meiotic nuclei are in pachytene. We found that homolog pairing occurs rapidly after karyogamy, that a 1 Mb chromosome does not associate more quickly than a 2.5 Mb chromosome, and that interstitial, single-copy sites can associate stably prior to nucleolar fusion. Analysis of two probes for the same pair of homologs revealed that by 4 h after karyogamy each chromosome examined was at least partially paired in all meiotic cells. In addition, these studies showed that chromatin condensation increases after pairing and that chromatin shows stable compaction at pachytene. Received: 4 January 1999; in revised form: 22 June 1999 / Accepted: 20 July 1999     

Inheritance of Chromosome-Length Polymorphisms in Coprinus Cinereus

We have investigated the inheritance of chromosome-length polymorphisms in the basidiomycete Coprinus cinereus. The electrophoretic karyotypes of interfertile strains of C. cinereus are strikingly different, and crosses between strains with different karyotypes yield progeny with chromosomes of new sizes. Repeated backcrossing of a mutant to one parent often stabilizes the mutant chromosome at a unique size; this then becomes a chromosome-length polymorphism marker for that mutant gene. A comparison of mutant strains, their wild-type progenitor, and backcrossed strains revealed that these marker chromosomes are not caused by the initial mutagenic treatment and are found only in progeny of crosses between strains with polymorphic chromosomes. Thus, they are most likely formed by meiotic recombination. For the rad12 gene, the marker chromosome can further recombine to become the size of the homolog of the backcross parent. For the rad3 gene, both ectopic and homologous recombination events are likely involved in the generation of the marker chromosomes. As predicted by a recombination model, a cross to a new wild-type parent can change the size of a mutant marker chromosome. Therefore, changes in chromosome length are a common and prominent feature of the genome of this sexual fungus, and a variety of karyotypes is tolerated by the organism.     

Multiple roles of Spo11 in meiotic chromosome behavior

Spo11, a type II topoisomerase, is likely to be required universally for initiation of meiotic recombination. However, a dichotomy exists between budding yeast and the animals Caenorhabditis elegans and Drosophila melanogaster with respect to additional roles of Spo11 in meiosis. In Saccharomyces cerevisiae, Spo11 is required for homolog pairing, as well as axial element (AE) and synaptonemal complex (SC) formation. All of these functions are Spo11 independent in C.elegans and D.melanogaster. We examined Spo11 function in a multicellular fungus, Coprinus cinereus. The C.cinereus spo11-1 mutant shows high levels of homolog pairing and occasionally forms full-length AEs, but no SC. In C.cinereus, Spo11 is also required for maintenance of meiotic chromosome condensation and proper spindle formation. Meiotic progression in spo11-1 is aberrant; late in meiosis basidia undergo programmed cell death (PCD). To our knowledge, this is the first example of meiotic PCD outside the animal kingdom. Ionizing radiation can partially rescue spo11-1 for both AE and SC formation and viable spore production, suggesting that the double-strand break function of Spo11 is conserved and is required for these functions.     

An mre11 mutant of Coprinus cinereus has defects in meiotic chromosome pairing, condensation and synapsis

The rad11 gene of the basidiomycete Coprinus cinereus is required for the completion of meiosis and for survival after gamma irradiation. We have cloned the rad11 gene and shown that it is a homolog of MRE11, a gene required for meiosis and DNA repair in numerous organisms. The expression of C. cinereus mre11 is induced during prophase I of meiosis and following gamma irradiation. The gene encodes a predicted polypeptide of 731 amino acids, and the mre11-1 (rad11-1) mutation is a single base pair change that results in a stop codon after amino acid 315. The mre11-1 mutant shows enhanced sensitivity to ionizing radiation, but no enhanced sensitivity to UV radiation. It shows a delay in fruitbody formation and a reduction in the number of mushrooms formed per dikaryon. The mre11-1 mutant also has several meiotic defects. Pachytene chromatin condensation is disrupted, and although some meiotic cells appear to achieve metaphase I condensation, no further meiotic progression is observed. The mre11-1 mutant also fails to undergo proper chromosome synapsis; neither axial elements nor mature synaptonemal complexes are complete. Finally, meiotic homolog pairing is reduced in the mre11-1 mutant. Thus, in C. cinereus, Mre11 is required for meiotic DNA metabolism.     

Pathway analysis of radiation-sensitive meiotic mutants ofCoprinus cinereus

We have isolated 37 radiation-sensitive mutants of the basidiomyceteCoprinus cinereus. Each mutation is recessive, and the collection defines at least ten complementation groups for survival of gamma irradiation. Four complementation groups define the genesrad3, rad9, rad11 andrad12, which are required both for survival of gamma irradiation and for meiosis. Mutants in each of these four groups fail to complete meiosis and produce mushrooms with greatly reduced numbers of viable spores. Propidium iodide staining of meiotic nuclei showed a characteristic terminal appearance for each mutant: few cells of any of the meiotic mutants progress beyond prophase I, and both condensation and fragmentation or dispersal of meiotic chromatin are frequently observed. Scanning electron micrographs showed that the meiotic mutants make varying numbers (0–6) of basidiospore initials and that few of these initials develop into mature spores. When initials are present they are always symmetrically arrayed on the basidium, regardless of initial number. In quantitative measurements of gamma ray sensitivity, double mutants of every tested combination ofrad3, rad9, rad11 andrad12 consistently showed the same gamma ray sensitivity as the more sensitive single mutant parent of the cross. Therefore, these four genes are in the same pathway for the repair of gamma radiation damage, and this pathway also represents one or more functions essential for meiosis.     

Repair Responses to DNA Damage: Enzymatic Pathways in E coli and Human Cells

Bacteria and eukaryotic cells employ a variety of enzymatic pathways to remove damage from DNA or to lessen its impact upon cellular functions. Most of these processes were discovered in Escherichia coli and have been most extensively analyzed in this organism because suitable mutants have been isolated and characterized. Analogous pathways have been inferred to exist in mammalian cells from the presence of enzyme activities similar to those known to be involved in repair in bacteria, from the analysis of events in cells treated with DNA damaging agents, and from the analysis of the few naturally occurring mutant cell types. Excision repair of pyrimidine dimers produced by UV in E coli is initiated by an incision event catalyzed by a complex composed of uvrA, uvrB, and uvrC gene products. Multiple exonuclease and polymerase activities are available for the subsequent excision and resynthesis steps. In addition to the constitutive pathway, which produces short patches of 20–30 nucleotides, an inducible excision repair process exists that produces much longer patches. This long patch pathway is controlled by the recA-lexA regulatory circuit and also requires the recF gene. It is apparently not responsible for UV-induced mutagenesis. However, the ability to perform inducible long patch repair correlates with enhanced bacterial survival and with a major component of the Weigle reactivation of bacteriophage with double-strand DNA genomes. Mammalian cells possess an excision repair pathway similar to the constitutive pathway in E coli. Although not as well understood, the incision event is at least as complex, and repair resynthesis produces patches of about the same size as the constitutive short patches. In mammalian cells, no patches comparable in size to those produced by the inducible pathway of E coli are observed. Repair in mammalian cells may be more complicated than in bacteria because of the structure of chromatin, which can affect both the distribution of DNA damage and its accessibility to repair enzymes. A coordinated alteration and reassembly of chromatin at sites of repair may be required. We have observed that the sensitivity of digestion by staphylococcal nuclease (SN) of newly synthesized repair patches resulting from excision of furocoumarin adducts changes with time in the same way as that of patches resulting from excision of pyrimidine dimers. Since furocoumarin adducts are formed only in the SN-sensitive linker DNA between nucleosome cores, this suggests that after repair resynthesis is completed, the nucleosome cores in the region of the repair event do not return exactly to their original positions. We have also studied excision repair of UV and chemical damage in the highly repeated 172 base pair α DNA sequence in African green monkey cells. In UV irradiated cells, the rate and extent of repair resynthesis in this sequence is similar to that in bulk DNA. However, in cells containing furocoumarin adducts, repair resynthesis in α DNA is only about 30% of that in bulk DNA. Since the frequency of adducts does not seem to be reduced in α DNA, it appears that certain adducts in this unique DNA may be less accessible to repair. Endonuclease V of bacteriophage T4 incises DNA at pyrimidine dimers by cleaving first the glycosylic bond between deoxyribose and the 5′ pyrimidine of the dimer and then the phosphodiester bond between the two pyrimidines. We have cloned the gene (denV) that codes for this enzyme and have demonstrated its expression in uvrA recA and uvrB recA cells of E coli. Because T4 endonuclease V can alleviate the excision repair deficiency of xeroderma pigmentosum when added to permeabilized cells or to isolated nuclei after UV irradiation, the cloned denV gene may ultimately be of value for analyzing DNA repair pathways in cultured human cells.