Recombination and hydroxyurea inhibition of DNA synthesis in yeast meiosis

Summary Hydroxyurea (HU) inhibits the premeiotic DNA replication and the meiotic events that follow, namely readiness, recombination commitment, haploidisation, sporulation commitment and ascus formation. Short incubations with HU (2–4 hrs) during the premeiotic replication (i.e. starting between 3 and 6.5 hrs in sporulation medium) allow the resumption of the replication at a normal rate following the removal of the drug. The other meiotic events are similarly delayed by the approximate length of the treatment. In these experiments, intragenic recombination in ade2 reached a higher level than in the controls (x1.3–2.0 in one pair of heteroalleles and x3.0–4.0 in another pair). The recombination response to short HU treatments was not observed for a pair of heteroalleles in ade2 that normally shows a high level of meiotic recombination (750 per 10⁶ cells), nor was the response observed in a pair of heteroalleles in lys2. HU treatments have almost no effect on sporulating cells from 8 hrs onwards. At 7–7.5 hrs the meiotic cells are very sensitive to the drug and even short treatments cause cell death and massive DNA degradation.     

Suppressor mutations (rin) that specifically suppress the recA+ dependence of stable DNA replication in Escherichia coli K-12

Summary The sdrA102 mutation confers upon cells the ability to replicate DNA in the absence of protein synthesis. This mutation was combined with the recA200 mutation, which renders the recA protein thermolabile, and had little effect on normal replication. However, the sdrA102 recA200 double mutant exhibited temperature-sensitive stable DNA replication: it replicated DNA continuously in the presence of chloramphenicol at 30°C, whereas at 42°C DNA replication ceased after the DNA content increased only 40–45%. Suppressor mutants (rin; recA-independent) capable of stable DNA replication at 42°C were isolated from the double mutant. The suppressor mutant retained all other recA ^– characteristics, i.e., deficient general recombination, severe UV-sensitivity, and incapability of prophage induction in lysogens. This indicates that the rin mutation specifically suppresses the recA ⁺ dependency of stable DNA replication. It is suggested that the recA ⁺ protein stabilizes a specific structure, similar to an intermediate in recombination, which may function in the initiation of stable DNA replication.     

Peculiarities of the antioxidant and radioprotective effects of hydrated C60 fullerene nanostuctures in vitro and in vivo

Aqueous solutions of highly stable supramolecular donor–acceptor complexes of chemically nonmodified pristine C₆₀ fullerene molecules with H₂O molecules (hydrated C₆₀ fullerene–C₆₀HyFn) and their labile nano-sized clusters were examined for their antioxidant effects on removal of hydroxyl radicals (OH) and protecting DNA against oxidative damage induced by ionizing radiation in vitro. The suppressing influence of C₆₀HyFn on the formation of OH-radicals in water exposed to X-rays at doses of 1–7 Gy was assessed by determination of oxidation levels of coumarin-3-carboxylic acid. C₆₀HyFn demonstrates apparent antiradical activity in vitro in the range of concentrations of 10⁻¹¹–10⁻⁶ M. Paradoxically, the OH-removing efficacy of C₆₀HyFn was in reverse correlation with fullerene concentration. It was hypothesized that the antiradical action of C₆₀HyFn in water medium generally is due to a “nonstoichiometric” mechanism, supposedly to a hydrated free radical recombination (self-neutralization), which is catalyzed by specific water structures ordered by C₆₀HyFn. With the use of 8-oxoguanine as a marker of oxidative damage to DNA, it has been demonstrated that C₆₀HyFn in concentrations of 10⁻⁷–10⁻⁶ M protects nucleic acids against radical-induced damage. The second part of the present study was aimed to evaluate the overall radioprotective efficacy of C₆₀HyFn in doses of 0.1 or 1 mg/kg b.w. injected intraperitoneally to mice either 1 h before or 15 min after lethal dose exposure of the X-ray (7 Gy) irradiation. Survival rate of the mice was observed at 30 day intervals after irradiation, while the weight gains of experimental animals were monitored as well. The most significant protective effect was demonstrated when 1 mg/kg dosage of C₆₀HyFn was administered before irradiation. The outcome of the substance testing is 15% survival rate of irradiated animals at 30 days of observation, and prevention of noticeable weight loss characteristic for radiation impact, versus unprotected control animals. In conclusion, results of the study obviate that the apparent protective action of C₆₀HyFn in vivo is determined by its considerable ability to decrease X-ray-generated reactive oxygen species. Based on the results and that neat C₆₀ is nontoxic, actually in the hydrated form, without side effects and with sufficient radioprotective effects in low doses, C₆₀HyFn may be considered as a novel antioxidant agent, which substantially diminishes the harmful effects of ionizing radiation.     

Properties of strains of Escherichia coli K12 carrying mutant lex and rec alleles

Summary Strains of Escherichia coli K12 have been constructed which carry the lex-3 mutation in combination with recA56, recB21, or recC22. The lex ^– recA ^–strain is equally as sensitive to ultraviolet light (UV) and ionizing radiation and as recombination-deficient as the corresponding lex ⁺ recA ^–strain, whereas the lex ^– recB ^–and lex ^– recC ^–strains are somewhat more sensitive to UV and ionizing radiation than the corresponding lex ^– rec ⁺strain and have approximately the same recombination deficiencies as the respective lex ⁺ recB ^–and Lex ⁺ recC ^–strains. When cultures of the lex ^– recB ^–and lex ^–recC^–strains are UV-irradiated, they degrade their DNA at the low rate characteristic of lex ⁺ recB ^–and lex ⁺ recC ^–single mutants, in contrast to the high rate of degradation seen with lex ^– rec ⁺single mutants.These results imply that the lex ⁺and recA ⁺products act in the same pathway of DNA repair and that both are needed to limit the DNA breakdown due to the recB ⁺/C⁺nuclease.     

Efficient gene targeting in the filamentous fungusAlternaria alternata

To characterize homologous recombination of transforming DNA in the filamentous fungusAlternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. TheA. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus.BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internalBRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1^–) transformants accounted for 30 to 80% of hygromycin B-resistant (Hy^R) transformants, correlating closely with the size of theBRM1 segment in the transforming DNA. Restriction enzyme digestion within theBRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of theBRM1 segments. Plasmids carrying bothBRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1^– transformants accounted for only 5% of Hy^R transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within theBRM1 segment was used, almost all transformants were Me1^–. These results indicate that homologous integration of circular molecules inA. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.     

Structure and organization of rhodophyte and chromophyte plastid genomes: implications for the ancestry of plastids

Summary Recombinational repair is the means by which DNA double-strand breaks (DSBs) are repaired in yeast. DNA divergence between chromosomes was shown previously to inhibit repair in diploid G1 cells, resulting in chromosome loss at low nonlethal doses of ionizing radiation. Furthermore, 15–20% divergence prevents meiotic recombination between individual pairs of Saccharomyces cerevisiae and S. carlsbergensis chromosomes in an otherwise S. cerevisiae background. Based on analysis of the efficiency of DSB-induced chromosome loss and direct genetic detection of intragenic recombination, we conclude that limited DSB recombinational repair can occur between homoeologous chromosomes. There is no difference in loss between a repair-proficient Pms⁺ strain and a mismatch repair mutant, pms1. Since DSB recombinational repair is tolerant of diverged DNAs, this type of repair could lead to novel genes and altered chromosomes. The sensitivity to DSB-induced loss of 11 individual yeast artificial chromosomes (YACs) containing mouse or human (chromosome 21 or HeLa) DNA was determined. Recombinational repair between a pair of homologous HeLa YACs appears as efficient as that between homologous yeast chromosomes in that there is no loss at low radiation doses. Single YACs exhibited considerable variation in response, although the response for individual YACs was highly reproducible. Based on the results with the yeast homoeologous chromosomes, we propose that the potential exists for intra- YAC recombinational repair between diverged repeat DNA and that the extent of repair is dependent upon the amount of repeat DNA and the degree of divergence. The sensitivity of YACs containing mammalian DNA to ionizing radiation-induced loss may thus be an indicator of the extent of repeat DNA.     

Segregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes

In Escherichia coli fermentation processes, a drastic drop in viable cell count as measured by the number of colony forming units per ml (c.f.u. ml^–1) is often observed. This phenomenon was investigated in a process for the production of the recombinant fusion protein, promegapoietin (PMP). After induction, the number of c.f.u. ml^–1 dropped to 10% of its maximum though the biomass concentration continued to increase. Flow cytometric analysis of viability and intracellular concentration of PMP showed that almost all cells were alive and contributed to the production. Thus, the drop in the number of c.f.u. ml^–1 probably reflects a loss of cell division capability rather than cell death.     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^– mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

Intramolecular homologous recombination of linearized plasmids in Escherichia coli K12

Summary The efficacy of linear DNA as a substrate for general homologous recombination was demonstrated using BamHI-linearized pKLC8.5, a plasmid that carries internal direct repeats flanking the unique BamHI site. An analogous plasmid, pKLC2.31, was used in a parallel and comparative study of intramolecular homologous recombination in circular DNA substrates. When the rec ⁺ wild-type strain, AB1157, and its isogenic rec ^– derivatives were transformed with linear pKLC8.5 DNA, intramolecular homologous recombination was independent of recA, recB, recN, recO and exonuclease III (xth-1) functions. Although the recBCsbcA and recBCsbcBC cells were both very recombination proficient, only linear but not circular DNA was used as substrate for intramolecular homologous recombination in the recBCsbcA cells. In both the recBCsbcA and recBCsbcBC genetic backgrounds, the recombination frequencies for linearized pKLC8.5 DNA were 100%. A notable difference between the two strains was that none of the recBCsbcA transformants obtained with circular pKLC8.5 DNA were Tc^s recombinants, whereas 11% of the corresponding recBCsbcBC transformants were Tc^s recombinants. The sbcB mutation was responsible for the recombination proficiency of the recBCsbcBC cells. Unlike the case in recBCsbcA cells, intramolecular homologous recombination of linear DNA in the recBCsbcBC cells was dependent on recA and recF as well as recN and recO gene functions, but was independent of recJ and reeL gene functions.     

Responses of Tradescantia stamen hairs and pollen to UV-B irradiation

Clones 02 and 4430 of Tradescantia were tested in field, greenhouse and controlled environment chambers as monitors for the potentially hazardous UV-B irradiation increase that could result from stratospheric ozone decrease. In addition to about 16 hr of solar emissions at about 2100 micro-einsteins·m⁻²·s⁻¹ (400–700 nm) and 15 hr at about 1800 micro-einsteins·m⁻²·s⁻¹ in the field and greenhouse, respectively, plants were given 7 hr of supplemental UV-B irradiation per day for 27 days. After the first 7 days of UV-B irradiation exposure, cumulative data were recorded for 20 days. Cuttings of Tradescantia plants in controlled-environment, exposed to 16 hr of simulated solar emission of about 800 micro-einsteins·m⁻²·s⁻¹ (400–700 nm), were also exposed to 10 hr of supplemental UV-B irradiation per day for 1 or 2 days. All plants were checked for somatic aberrations (color changes in the flower petals and stamen hairs), number of hairs per stamen, and cells per hair. Pollen germination and pollen tube growth were noted after a 90-min UV-B irradiation period.Somatic aberrations occurred infrequently in the petals and were judged unreliable criteria for use in monitoring enhanced UV-B irradiation environments. The number of aberrant events within stamen hairs, however, was significantly increased by the UV-B irradiation treatments. while pollen germination and pollen tube growth were significantly reduced. These data indicate that color changes in stamen hairs and pollen viability are useful criteria for monitoring UV-B irradiation changes.     

Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168

Summary We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB: : cat, leuB ^–, and pro: : neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB ⁺ pro⁺)(leuB: : cat pro ⁺) (leuB ^– pro: : neo)(leuB ^– pro ⁺) resulted in a leuB ^– single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD ^– and pro ^–, as well as the double mutation leuB ^– pro ^– can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.     

Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae

Summary DNA fragments carrying the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae were isolated by complementing a UV-sensitive recA ^– Escherichia coli strain. Sequence analysis revealed that the coding region of the R. meliloti recA gene consists of 1044 by coding for 348 amino acids whereas the coding region of the R. leguminosarum bv. viciae recA gene has 1053 bp specifying 351 amino acids. The R. meliloti and R. leguminosarum bv. viciae recA genes show 84.8% homology at the DNA sequence level and of 90.1% at the amino acid sequence level. recA ^– mutant strains of both Rhizobium species were constructed by inserting a gentamicin resistance cassette into the respective recA gene. The resulting recA mutants exhibited an increased sensitivity to UV irradiation, were impaired in their ability to perform homologous recombination and showed a slightly reduced growth rate when compared with the respective wild-type strains. The Rhizobium recA strains did not have altered symbiotic nitrogen fixation capacity. Therefore, they represent ideal candidates for release experiments with impaired strains.The accession numbers: X59956 R. LEGUMINOSARUM REC A ALAS-DNA; X59957 R. MELITOTI REC A ALAS-DNA     

Reduced digestibility of UV-B stressed and nutrient-limited algae by Daphnia magna

Daphnia magna was fed the green alga Selenastrum capricornutum, cultured under four different growth conditions: (1) phosphorus limitation, (2) nitrogen limitation, (3) UV-B irradiation, and (4) no nutrient limitation, no UV-B irradiation. Contrary to non-limited algal cells, nutrient-limited cells were not efficiently assimilated. Especially, P-limited cells passed through the gut mostly intact, while N-limited cells were partly assimilated. Also, algae exposed to moderate doses of UV-B radiation (0.3 mW cm^–2 of UV₃₁₂) were less efficiently assimilated after being grazed. Digestibility of the algae decreased with increased UV-B exposure time. Nutrient-limited and UV-B stressed algal cells increased in volume and became granular in appearance. These changes in the algal cells, combined with changed cell wall properties, most probably reduced their digestibility.     

Atm-dependent interactions of a mammalian Chk1 homolog with meiotic chromosomes

Background: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest.Results: We have identified structural homologs of yeast Chk1 in human and mouse. Chk1^Hu/Mo has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions. Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22–23, a region marked by frequent deletions and loss of heterozygosity in human tumors.Conclusions: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22–23 indicates that the CHK1 gene is a candidate tumor suppressor gene.     

Recombination and mating-type switching in a ligase-defective mutant of Schizosaccharomyces pombe

Summary The ligase-defective cdc17-L16 mutant of Schizosaccharomyces pombe var. pombe was tested for genetic recombination and mating-type switching. Mitotic recombination was studied in both haploid and heteroallelic diploid cells. Cells carrying a heteroallelic ade6 duplication constructed by Schuchert and Kohli were tested for ectopic genetic recombination. We have found that cdc17-L16 is a mitotic hyper-rec mutant, as it increases the instability of the duplication by a factor of about 6 even at the permissive temperature of 23° C. In diploid cells, the enhancement of recombination rates detected was to that of cdc17 ⁺ cells. The temperature-sensitive cell cycle defect is also associated with a reduced level of mating and sporulation but does not significantly affect mating-type switching and intragenic meiotic recombination. It is supposed that the mitotic hyper-rec phenotype is a secondary result of insufficient repair of DNA breaks, while the lack of influence of the reduced ligase activity on the latter two processes might be attributed to their peculiar initiation mechanisms.     

Dependence of DNA dark repair on protein synthesis in Escherichia coli.

Summary We investigated the influence of aminoacidless treatments applied prior and after UV irradiation on survival, dimer excision, postirradiation DNA degradation, DNA synthesis and sedimentation profiles of parental DNA ofE. coli B/r Hcr⁺ cells. In dependence on the treatment applied, the fluence 50 J/m² yielded distinctly different fractions of survivors within 0,03–85%. In all cases dimers were completely excised. The rate of DNA degradation was similar during a 30–40 min period after UV during which the bulk of dimers was excised. Degradation ceased, however, earlier in the prestarved cells than in exponentially growing ones; it was prolonged by aminoacidless postincubation. Sedimentation profiles of parental DNA did not differ during the whole period of dimer excision. In cells DNA synthesis was not restored for several hours after addition of amino acids. In cells addition of amino acids resulted in a fast resumption of DNA synthesis. We conclude that removal of dimers and repair of gaps were similar in all cases. We believe that aminoacidless treatments influence production and repair of damage to the sites of DNA replication. The treatment appears to prevent this damage when applied before UV irradiation, but interferes with its restoration when applied after UV irradiation. Consequently, the former treatment increases survival of cells while the latter produces an opposite effects.     

Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25°C. Replacement of K⁺ with Na⁺ effectively prevented the lysis by SCN⁻. K⁺ salts of NO₃⁻, Br⁻, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN⁻ than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN⁻ is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na⁺ observed here was considered to be manifested by specific interactions of Na⁺ with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.     

Hyper-resistance of meiotic cells to radiation due to a strong expression of a single recA-like gene in Caenorhabditis elegans

Sensitivity of meiotic cells to DNA damaging agents is little understood. We have demonstrated that the meiotic pachytene nuclei in the Caenorhabditis elegans gonad are hyper-resistant to X-ray irradiation, but not to UV irradiation, whereas the early embryonic cells after fertilization and the full grown oocytes are not. The Ce-rdh-1 gene [RAD51, DMC1 (LIM15), homolog 1 or Ce-rad-51], which is essential for the meiotic recombination, is the only bacterial recA-like gene in the nematode genome, and is strongly expressed in the meiotic cells. Following silencing of the Ce-rdh-1 gene by RNA interference, the meiotic cells become more sensitive to X-ray irradiation than the early embryonic cells. This is the first report that meiotic cells are hyper-resistant to DNA strand breaks due to the high level of expression of the enzyme(s) involved in meiotic homologous recombination.     

Hyper-recombination in dam mutants of Escherichia coli K-12

Summary F-prime heterogenotes of dam-3 bacteria segregate F-prime homogenotes at a frequency 30–200 times higher than the isogenic dam ⁺ strain. A hyperrecombination mutant which shows increased recombination between chromosomal duplications was characterized as a dam mutant. The dam-3 allele causes a reduction in linkage of proximal unselected markers in transconjugants and increases the recombination frequency between a pair of closely linked markers. It is concluded that dam mutations confer a hyperrecombination phenotype to the cell.     

Developmental genetics of a temperature-sensitive RNA polymerase II mutation in Drosophila melanogaster

Summary Purified RNA polymerase II (RNA nucleotidyl-transferase; EC 2.7.7.6) extracted from flies possessing lesions in the Ultrabithorax-like (Ubl) locus of Drosophila melanogaster has altered activity in vitro (Greenleaf et al. 1979, 1980; Coulter and Greenleaf 1982). This strongly suggests that the Ubl locus encodes a subunit of RNA polymerase II. Ethyl methanesulfonate was used to induce a temperature-sensitive mutation in this locus. Flies either homozygous or hemizygous for this new X–linked mutation (Ubl ^ts) display viability comparable to that of wild-type flies at 22° C but are lethal at 29° C. The temperature-sensitive period for Ubl ^ts flies is between gastrulation (6 h, 29° C) and pupation (9–10 days, 22° C). Zygotes shifted from 22° C to 29° C die at either the late embryonic or first larval instar stage while temperature shifts of second and third instar larvae result in the lethal phase occurring at the pupal stage. Most pupae shifted from 22° C to 29° C undergo metamorphosis and eclose as adults. Adults are viable if placed at 29° C; however, all females and some males become sterile if maintained at this temperature.Somatic recombination was used to induce clones homozygous for a null allele of Ubl at different stages of development. Clones of this null allele appear to be cell lethal indicating that the Ubl ⁺ gene product is required at all stages of development. The viability of Ubl ^ts pupae and adults at 29° C may result from only a partial reduction in activity caused by the mutation at this nonpermissive temperature.