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To elucidate the function of metazoan B-type lamins during development, new null mutations of the Drosophila B-type lamin gene, lamDm0, were analyzed in parallel with the misgsz18 mutation, a lamDm0 allele reported previously. Although in all these mutants, lamin Dm0 protein was undetectable in neuroblasts and imaginal disc cells from the second instar larval stage onward, cells continued to proliferate. In contrast to the embryonic lethality of another Drosophila lamDm0 allele, lamPM15, reported previously, lethality did not occur until late pupal stages. Chromosomal structure and the overall nuclear shape remained normal even at these late pupal stages, although obviously abnormal nuclear pore complex distribution was observed concomitant with the loss of lamin Dm0 protein. Compensating expression of lamin C was not induced in the absence of lamin Dm0. Thus, no lamin-containing nuclear structures were found in proliferating larval neuroblasts. We did find that developmental abnormalities appeared in specific organs during the late pupal stage, preceding lethality. Surprisingly, coordinated size increase (hypertrophy) of the ventriculus was observed accompanied by cell division and muscle layer formation. Hypertrophy of the ventriculus correlated with a decrease in ecdysteroid hormone receptor B1 (EcRB1) protein, and furthermore could be suppressed by a heat-inducible EcRB1 transgene. In contrast, both gonadal and CNS tissues exhibited underdevelopment.

Proliferating cell nuclear antigen (PCNA), a protein intimately involved in both replication and repair, has been identified in eukaryotes at all levels of evolution. Is primary sequence, Drosophila melanogaster PCNA is 73% identical to mammalian PCNA. Moreover, it is able to substitute for mammalian PCNA in at least one intricate cell-free replication assay. Mutations in the gene for Drosophila PCNA, including some that are temperature sensitive, have been reported. Procedures are described for the biochemical purification of wild-type PCNA from a population of 6- to 18-h-old Drosophila embryos. Procedures were also developed for purification of unmodified wild-type Drosophila PCNA after induction of expression in Escherichia coli. An NH(2)-terminally His-tagged but otherwise wild-type Drosophila PCNA, as well as mutant His-tagged PCNA, were also engineered and purified to apparent homogeneity. Finally, an in situ polyacrylamide gel technique allows DNA polymerase assays to be performed on portions of single adults as well as single Drosophila embryos. This assay should tremendously facilitate systematic genetic studies of metazoan replication and repair.

A proliferating cell nuclear antigen (PCNA)-dependent complex, detectable after nondenaturing polyacrylamide gel electrophoresis, is formed between calf thymus DNA polymerase delta (pol delta) and synthetic oligonucleotide template-primers containing a mispaired nucleotide at the 3'-terminal position of the primer. This complex is indistinguishable in composition from that formed with a fully base paired template-primer. Extension of a mispaired primer terminus is a component of DNA polymerase fidelity. The fidelity of pol delta on synthetic oligonucleotide template-primers was compared with and without its specific processivity factor, PCNA. In the absence of PCNA, pol delta misincorporates less than one nucleotide for every 100,000 nucleotides incorporated correctly. Addition of PCNA to reactions reduces fidelity by at least 27-fold. PCNA also confers upon pol delta, the ability to incorporate (and/or not excise) the dTTP analog, 2'-deoxythymidine-5'-O-(alpha-phosphonomethyl)-beta, gamma-diphosphate. A model is proposed whereby the increased stability (decreased off-rate) of the pol delta.template-primer complex in the presence of PCNA facilitates unfavorable events catalyzed by pol delta. This model suggests an explicit mechanistic requirement for the intrinsic 3'-5'-exonuclease of pol delta.

Most DNA topoisomerase II (topo II) in cell-free extracts of 0-2-h old Drosophila embryos appears to be nonnuclear and remains in the supernatant after low-speed centrifugation (10,000 g). Virtually all of this apparently soluble topo II is particulate with a sedimentation coefficient of 67 S. Similar topo II-containing particles were detected in Drosophila Kc tissue culture cells, 16-19-h old embryos and extracts of progesterone-matured oocytes from Xenopus. Drosophila topo II-containing particles were insensitive to EDTA, Triton X-100 and DNase I, but could be disrupted by incubation with 0.3 M NaCl or RNase A. After either disruptive treatment, topo II sedimented at 9 S. topo II-containing particles were also sensitive to micrococcal nuclease. Results of chemical cross-linking corroborated those obtained by centrifugation. Immunoblot analyses demonstrated that topo II-containing particles lacked significant amounts of lamin, nuclear pore complex protein gp210, proliferating cell nuclear antigen, RNA polymerase II subunits, histones, coilin, and nucleolin. Northern blot analyses demonstrated that topo II-containing particles lacked U RNA. Thus, current data support the notion that nonnuclear Drosophila topo II-containing particles are composed largely of topo II and an unknown RNA molecule(s).

To elucidate the function of metazoan B-type lamins during development, new null mutations of the Drosophila B-type lamin gene, lamDm(0), were analyzed in parallel with the misg(sz18) mutation, a lamDm(0) allele reported previously. Although in all these mutants, lamin Dm(0) protein was undetectable in neuroblasts and imaginal disc cells from the second instar larval stage onward, cells continued to proliferate. In contrast to the embryonic lethality of another Drosophila lamDm(0) allele, lam(PM15), reported previously, lethality did not occur until late pupal stages. Chromosomal structure and the overall nuclear shape remained normal even at these late pupal stages, although obviously abnormal nuclear pore complex distribution was observed concomitant with the loss of lamin Dm(0) protein. Compensating expression of lamin C was not induced in the absence of lamin Dm(0). Thus, no lamin-containing nuclear structures were found in proliferating larval neuroblasts. We did find that developmental abnormalities appeared in specific organs during the late pupal stage, preceding lethality. Surprisingly, coordinated size increase (hypertrophy) of the ventriculus was observed accompanied by cell division and muscle layer formation. Hypertrophy of the ventriculus correlated with a decrease in ecdysteroid hormone receptor B1 (EcRB1) protein, and furthermore could be suppressed by a heat-inducible EcRB1 transgene. In contrast, both gonadal and CNS tissues exhibited underdevelopment.

mus 308 designates one of over 30 mutagen sensitivity loci found in Drosophila. It is predicted to code for a 229-kDa polypeptide. Published sequence analyses of others indicate that this polypeptide would have helicase motifs near its N-terminus, and similarities to bacterial DNA polymerase I-like enzymes near its C-terminus. In our studies, two different and highly specific antibodies were prepared and used for identification as well as characterization of the mus 308 gene product. Western blot analyses reveal a single reactive polypeptide in both ovaries and embryos as well as in two Drosophila embryo tissue culture cell lines; it is nearly absent in homozygous mus 308 mutants. This polypeptide is about 229 kDa in size, and indirect immunofluorescence shows that the mus 308 gene product localizes throughout nuclei in wild-type cells but appears to be absent in a mus 308 mutant. Immunoblot analyses throughout development suggest greatest abundance at the end of embryogenesis, immediately before hatching of first instar larvae. They also showed a smaller ( approximately 100 kDa) antigenically and genetically related polypeptide found only in adult males. Immunoprecipitation, a highly effective method of specific purification, suggests that the mus 308 protein has DNA polymerase activity that is NEM-sensitive but largely aphidicolin-resistant. In addition, the immunoprecipitated material has DNA-dependent ATPase but lacks detectable helicase.

Barrier-to-autointegration factor (BAF) is potentially a DNA-bridging protein, which directly associates with inner nuclear membrane proteins carrying LEM domains. These features point to a key role in regulation of nuclear function and organization, dependent on interactions between the nuclear envelope and chromatin. To understand the functions of BAF in vivo, Drosophila baf null mutants generated by P-element-mediated imprecise excision were analyzed. Homozygous null mutants showed a typical mitotic mutant phenotype: lethality at the larval-pupal transition with small brains and missing imaginal discs. Mitotic figures were decreased but a defined anaphase defect as reported for C. elegans RNAi experiments was not observed in these small brains, suggesting a different phase or phases of cell cycle arrest. Specific abnormalities in interphase nuclear structure were frequently found upon electron microscopic examination of baf null mutants, with partial clumping of chromatin and convolution of nuclear shape. At the light microscopic level, grossly aberrant nuclear lamina structure and B-type lamin distribution correlated well with the loss of detectable amounts of BAF protein from nuclei. Together, these data represent evidence of BAF's anticipated function in mediating interactions between the nuclear envelope and interphase chromosomes. We thus conclude that BAF plays essential roles in nuclear organization and that these BAF functions are required in both M phase and interphase of the cell cycle.

CRP1, a Drosophila nuclear protein that can catalyze decondensation of demembranated Xenopus sperm chromatin was cloned and its primary structure was deduced from cDNA sequence. Alignment of deduced amino acid sequence with published sequences of other proteins revealed strong homologies to Xenopus nucleoplasmin and NO38. CRP1 is encoded by one or several closely related genes found at a single locus, position 99A on the right arm of chromosome 3. CRP1 mRNA is expressed throughout Drosophila development; it is highest during oogenesis and early embryogenesis. mRNA levels correlate closely with levels of protein expression measured previously. Results of chemical crosslinking indicate that CRP1 is either tetrameric or pentameric; similar ambiguity was revealed by direct visualization using scanning transmission electron microscopy. Consistent with previously published results, parallel crosslinking studies of Xenopus nucleoplasmin suggested a pentameric structure. Scanning transmission electron microscopic examination after negative staining revealed that CRP1 and Xenopus nucleoplasmin are morphologically similar. CRP1 is able to substitute for nucleoplasmin in Xenopus egg extract-mediated sperm chromatin decondensation. In vitro, CRP1-induced decondensation is accompanied by direct binding of CRP1 to chromatin.

Three direct assays, polyacrylamide gel electrophoresis-band mobility shift, agarose gel electrophoresis-band mobility shift, and nitrocellulose filter binding, were established to study complexes formed among mammalian DNA polymerase delta (pol delta), proliferating cell nuclear antigen (PCNA), and synthetic oligonucleotide template-primers. In all contexts, complex formation requires simultaneous presence of pol delta, PCNA, and template-primer. Moreover, we showed in one such assay that the complex formed contains each molecular component. Nuclease protection experiments demonstrate that complex formation protects template from degradation by DNase I. The mass determined for the pol delta.PCNA.template-primer complex was about 267 kDa, consistent with the participation of one molecule of pol delta, two or three molecules of PCNA and one molecule of template-primer. PCNA alone behaved as a trimer (mass determined to be about 87 kDa). Complex could be manipulated enzymologically. Measurement of off rates demonstrates directly that PCNA stabilizes the pol delta.template-primer complex.

Background We and others have shown four distinct and presumably related effects of mammalian proliferating cell nuclear antigen (PCNA) on DNA synthesis catalyzed by mammalian DNA polymerase δ(pol δ). In the presence of homologous PCNA, pol δ exhibits 1) increased absolute activity; 2) increased processivity of DNA synthesis; 3) stable binding of synthetic oligonucleotide template-primers (t1/2 of the pol δ•PCNA•template-primer complex ≥2.5 h); and 4) enhanced synthesis of DNA opposite and beyond template base lesions. This last effect is potentially mutagenic in vivo. Biochemical studies performed in parallel with in vivo genetic analyses, would represent an extremely powerful approach to investigate further, both DNA replication and repair in eukaryotes.

Aided and free recall procedures found that a random sample remembered videotex news stories better than newscaster‐presented stories. Graphics helped recall of a story's topic more than its details. Nonredundant crawls hindered aided recall of both newscaster and videotex stories. A crawl was better remembered when it accompanied newscaster stories than videotex stories. Videotex stories with a crawl were better remembered than newscaster stories with a crawl.