Progression of meiotic DNA replication is modulated by interchromosomal interaction proteins, negatively by Spo11p and positively by Rec8p.

Spo11p is a key mediator of interhomolog interactions during meiosis. Deletion of the SPO11 gene decreases the length of S phase by approximately 25%. Rec8p is a key coordinator of meiotic interhomolog and intersister interactions. Deletion of the REC8 gene increases S-phase length, by approximately 10% in wild-type and approximately 30% in a spo11Delta background. Thus, the progression of DNA replication is modulated by interchromosomal interaction proteins. The spo11-Y135F DSB (double strand break) catalysis-defective mutant is normal for S-phase modulation and DSB-independent homolog pairing but is defective for later events, formation of DSBs, and synaptonemal complexes. Thus, earlier and later functions of Spo11 are defined. We propose that meiotic S-phase progression is linked directly to development of specific chromosomal features required for meiotic interhomolog interactions and that this feedback process is built upon a more fundamental mechanism, common to all cell types, by which S-phase progression is coupled to development of nascent intersister connections and/or related aspects of chromosome morphogenesis. Roles for Rec8 and/or Spo11 in progression through other stages of meiosis are also revealed.

Somatic pairing of homologs in budding yeast: existence and modulation.

FISH analysis of well-spread chromosomes reveals that homologs are paired in vegetatively growing budding yeast diploid cells, via multiple interstitial interactions, and independent of recA homologs and mating type heterozygosity. Pairing is present during G1 and G2, and in cells arrested at G1 by mating pheromone, but is disrupted during S phase. Thus, somatic pairing is qualitatively analogous to premeiotic and early meiotic pairing. S-phase pairing disruption occurs by a complex intranuclear program involving regional, nucleus-wide, and temporal determinants. Pairing is also disrupted in two G2-arrest conditions (cdc13ts and nocodazole). Together these findings suggest that cell cycle signals may provoke pairing disruption by modulating underlying chromosome and/or chromatin structure. Whether the cell chooses to disrupt pairing contacts or not (e.g., S phase and G2 arrest, but not G1 arrest or normal G1 or G2), could be dictated by functional considerations involving homolog/sister discrimination.

Meiotic cells monitor the status of the interhomolog recombination complex.

During meiosis, mutations that cause defects at intermediate stages in the recombination process confer arrest at the end of prophase (e.g., pachytene). In yeast, mutations of this type include rad50S, dmc1, rad51, and zip1. Rad50 is likely part of a recombination initiation complex. DMC1, RAD51, and ZIP1 encode two RecA homologs and a synaptonemal complex protein, respectively. We report here the effects of mutations in two other (meiosis-specific) genes, RED1 and MEK1/MRE4, that encode a chromosome structure component and a protein kinase, respectively. A red1 or mek1/mre4 mutation alleviates completely rad50S, dmc1, rad51, and zip1 arrest. Furthermore, the red1 and mek1/mre4 mutations define a unique, previously unrecognized aspect of recombination imposed very early in the process, during DSB formation. Finally, the red1 and mek1/mre4 mutations appear to alleviate prophase arrest directly rather than by eliminating, or permitting bypass of, the rad50S, dmc1, rad51, or zip1 defects. These and other observations suggest that a meiosis-specific regulatory surveillance process monitors the status of the protein/DNA interhomolog recombination machinery as an integral entity, in its proper chromosomal context, and dependent upon its appropriate Red1 and Mek1/Mre4-promoted development. We speculate that a properly developed recombination complex emits an inhibitory signal to delay progression of meiotic cells out of prophase until or unless the recombination process has progressed, at least past certain critical steps, and perhaps to completion.

An N-terminal deletion mutant of simian virus 40 (SV40) large T antigen oligomerizes incorrectly on SV40 DNA but retains the ability to bind to DNA polymerase alpha and replicate SV40 DNA in vitro.

A peptide encompassing the N-terminal 82 amino acids of simian virus 40 (SV40) large T antigen was previously shown to bind to the large subunit of DNA polymerase alpha-primase (I. Dornreiter, A. Höss, A. K. Arthur, and E. Fanning, EMBO J. 9:3329-3336, 1990). We report here that a mutant T antigen, T83-708, lacking residues 2 to 82 retained the ability to bind to DNA polymerase alpha-primase, implying that it carries a second binding site for DNA polymerase alpha-primase. The mutant protein also retained ATPase, helicase, and SV40 origin DNA-binding activity. However, its SV40 DNA replication activity in vitro was reduced compared with that of wild-type protein. The reduction in replication activity was accompanied by a lower DNA-binding affinity to SV40 origin sequences and aberrant oligomerization on viral origin DNA. Thus, the first 82 residues of SV40 T antigen are not strictly required for its interaction with DNA polymerase alpha-primase or for DNA replication function but may play a role in correct hexamer assembly and efficient DNA binding at the origin.

Chromosome pairing via multiple interstitial interactions before and during meiosis in yeast.

Fluorescence in situ hybridization analysis reveals that homologous chromosomes are paired in yeast cells about to enter meiosis. Pairing involves multiple interstitial interactions, one per approximately 65 kb. These observations exclude several classes of models for somatic/premeiotic pairing. The number of t = 0 pairing interactions is about the same as the number of subsequent meiotic recombination events. As cells enter meiosis, pairing disappears concomitant with DNA replication and then reappears, independent of synaptonemal complex. Mutant phenotypes suggest that formation of an individual meiotic pairing connection does not require a meiosis-specific double-stranded break (DSB). Mutants defective in recombination before or after DSBs exhibit pairing defects. These and other observations can be united by a model in which premeiotic pairing and early meiotic pairing occur by closely related paranemic DNA-DNA interactions between intact duplexes, with early meiotic interactions subsequently converted directly to plectonemic recombination intermediates via DSBs.

Potential advantages of unstable interactions for pairing of chromosomes in meiotic, somatic, and premeiotic cells.

Many different aspects of chromosome pairing, meiotic and/or somatic, can be explained conveniently if the interactions between homologous chromosomes are unstable. Initial pairing interactions should involve very unstable contacts. Such interactions could go a long way toward bringing each pair of homologous chromosomes into a joint domain, free of ectopic associations and random entanglements with other chromosomes, and in a topologically acceptable relationship to the domains of other chromosome pairs. More generally, colocalization into topologically acceptable domains could be a useful way of defining the existence of "order" at early stages in pairing; this definition requires that chromosomes have in some way recognized and interacted with one another but does not require that they necessarily be in close apposition and/or that they be aligned along their entire lengths. At later stages, interactions between pairing chromosomes could be unstable but still reversible, either intrinsically or due to an active cell-directed process. Transient homologous interactions could also contribute to maintaining colocalization between homologous chromosomes through DNA replication.

A new biaspheric contact lens for severe astigmatism following penetrating keratoplasty.

We conducted a study of 26 consecutive post penetrating keratoplasty patients (32 eyes) fit with a Boston Envision biaspheric rigid gas permeable contact lens with limited parameters. The lens was successfully worn by 81% of those fit. Eighty-nine percent of patients' eyes had visual acuity equal to or better than their best possible spectacle acuity, and lens related complications were minimal.

Specific mutation of a regulatory site within the ATP-binding region of simian virus 40 large T antigen.

In an attempt to distinguish simian virus 40 (SV40) large T antigen (T) binding to ATP from hydrolysis, specific mutations were made in the ATP-binding site of T according to our model for the site (M. K. Bradley, T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster, Proc. Natl. Acad. Sci. USA 84:4026-4030, 1987). Two acidic residues predicted to make contact with the magnesium phosphate were changed to alanines. The mutated T gene was completely defective for viral DNA synthesis and for virion production, and it was dominant defective for viral DNA replication. The defective T gene encoded a stable product (2905T) that oncogenically transformed mouse cell lines. 2905T, immunoprecipitated from transformed-cell extracts, bound SV40 origin DNA specifically and, surprisingly, it was active as an ATPase. A recombinant baculovirus was constructed for the production and purification of the mutant protein for detailed biochemical analyses. 2905T had only 10% of the ATPase and helicase of wild-type T. The Km of 2905T for ATP in ATPase assays was the same as the Km of wild-type T. ATP activated the ATPase activity of wild-type T, but not of 2905T. As tested by gel bandshift assay, 2905T bound to SV40 origin DNA and to individual sites I and II with affinities similar to that of the wild type. However, ATP did not modulate the DNA-binding activity of mutant T to site II. Therefore, this mutation in the ATP-binding site in T resulted in defects in the interaction between the protein and ATP that appeared to be responsible for the determination of the active state of T for DNA binding versus ATPase.

Stimulation of the human heat shock protein 70 promoter in vitro by simian virus 40 large T antigen.

Simian virus 40 large tumor (T) antigen stimulates transcription from the SV40 late promoter and some cellular genes. We report here the novel finding that purified T antigen preferentially stimulates transcription from the human heat shock protein 70 promoter in an in vitro transcription system. T antigen is thus capable of stimulating transcription by a process that does not require synthesis of other proteins and that may involve a direct interaction with preexisting cellular factors.

Effects of two nonsteroidal anti-inflammatory drugs, indomethacin and oxaprozin, on the kidney.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been found to cause sodium retention and to decrease glomerular filtration rate (GFR). We studied the effects of two such drugs, indomethacin and oxaprozin, a new propionic acid derivative, on renal function of awake, normal human subjects during sustained water diuresis. Although neither drug had a long-term effect on GFR or sodium clearance (CNa), indomethacin (six subjects) but not oxaprozin (seven subjects) transiently reduced GFR and CNa. Given over the short term, oxaprozin caused a reduction in GFR from 113.7 +/- 5.7 to 99.8 +/- 4.7 ml/min (p < 0.01) and CNa from 0.84 +/- 0.07 to 0.61 +/- 0.08 ml/min (p < 0.005). The results were much the same when an additional dose of indomethacin was given to subjects who had been receiving the drug for a week. Inference from clearance data at a time when urinary osmolality (Uosm) remained constant but urine flow per GFR (V/GFR) fell suggests that both drugs stimulated proximal tubular sodium and fluid resorption. Both suppressed renin and aldosterone levels comparably and reduced potassium excretion transiently, but only indomethacin caused a sustained change in serum potassium concentration; serum potassium rose from 4.32 +/- 0.10 to 4.56 +/- 0.11 mEq/l (p < 0.05) after 1 wk. These disparate findings suggest that prostaglandin synthesis inhibition may not be the sole mechanism of action of NSAIDs.