Drosophila Kc Cells Research Articles

Loss of drug-stimulated topoisomerase II DNA breaks in living cells is different at two unrelated loci.

Topoisomerase II (top2) has been implicated in the initial steps of chromosomal translocations leading to leukemias and lymphomas, since it can generate DNA cleavage. To evaluate the effects of chromatin structure on enzyme-mediated cleavage, we determined the kinetics of loss of double-stranded DNA breaks stimulated by top2 poisons in Drosophila melanogaster Kc cells at two genomic regions that differ in chromatin structure. Moreover, cleavage loss was determined at 25 degrees C as well as after heat shock. Kinetics were dependent on the poison, nevertheless, loss rate overall was slow at the histone gene cluster, an active chromatin domain. At the repressed satellite III DNA, loss of cleavage was much faster and complete after 5 min in drug-free medium. In addition, differences were noted among sites that were closely spaced and equally intense. Following heat shock at 37 degrees C, we observed reduced cleavage levels and faster loss of breaks at the histone gene cluster. In vitro reversal could only partially explain the in vivo kinetics. Thus, the chromatin context of DNA breaks might play a role in the loss of top2 DNA breaks. The present findings suggest that irreversible cuts may more likely occur in active than silent loci.

The RRM Protein NonA from Drosophila Forms a Complex with the RRM Proteins Hrb87F and S5 and the Zn Finger Protein PEP on hnRNA

The RRM protein NonA, an ubiquitous nuclear protein present in puffs on polytene chromosomes, has been immunopurified as a RNA–protein complex from Drosophila Kc cells. Three other proteins present in the complex have been identified: X4/PEP (protein on ecdysone puffs), a 100-kDa zinc finger RNA-binding protein; the 70-kDa S5 protein, an as yet uncharacterized RNA-binding protein; and P11/Hrb87F, a 38-kDa RRM protein homologous to hnRNP protein A1 from mammals. Monoclonal antibodies against any of the protein components coprecipitate all four proteins although at different ratios. NonA does not coprecipitate with the hrp40 hnRNP proteins and immunolocalizes in a pattern distinct of major hnRNP proteins. Like NonA, X4/PEP, S5, and P11/Hrb87F are present on active sites on polytene chromosomes. The precipitated NonA complex is enriched for certain protein encoding RNAs, notably, histone H3 and H4 RNA.

A Binding Site for the Transcription Factor Grainyhead/Nuclear Transcription Factor-1 Contributes to Regulation of theDrosophila Proliferating Cell Nuclear Antigen Gene Promoter

The Drosophila proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including upstream regulatory element (URE), DNA replication-related element, E2F recognition sites, and three common regulatory factor for DNA replication and DNA replication-related element-binding factor genes recognition sites. In nuclear extracts of Drosophila embryos, we detected a protein factor, the URE-binding factor (UREF), that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE. Analyses in Drosophila Kc cells and transgenic flies revealed that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies. A yeast one-hybrid screen using URE as a bait allowed isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (GRH/NTF-1). The nucleotide sequence required for binding to GRH was indistinguishable from that for UREF detected in embryo nuclear extracts. Furthermore, a specific antibody to GRH reacted with UREF in embryo nuclear extracts. From these results we conclude that GRH is identical to UREF. Although GRH has been thought to be involved in regulation of differentiation-related genes, this study demonstrates, for the first time, involvement of a GRH-binding site in regulation of the DNA replication-related proliferating cell nuclear antigen gene.

Genomic sites of topoisomerase II activity determined by comparing DNA breakage enhanced by three distinct poisons

To define the sites of topoisomerase II activity in two genomic regions of Drosophila melanogaster Kc cells, we have investigated in vivo DNA cleavage sites stimulated by three poisons with diverse sequence specificity, clerocidin, VM-26 and dh-EPI (an anthracycline analog). DNA cleavage was studied by PFGE (pulse-field gel electrophoresis), standard gel electrophoresis, and by genomic primer extension. Poisons stimulated specific intensity patterns of cleavage in the two genomic regions studied. At the centromeric satellite III DNA, fragments of about 270–310 and 385–430 kb could be detected specifically after treatment with clerocidin, suggesting a complex DNA loop organisation, which may correspond with a centromere-specific higher-order chromatin structure. Clerocidin-dependent DNA fragmentation was detectable by PFGE but not by standard agarose gel electrophoresis; while VM-26-dependent cleavage was detected with either method, dh-EPI was ineffective at this locus. Thus, clerocidin DNA cleavage sites were rarer than those of VM-26 at the satellite locus. In the histone H2A-H2B intergenic region, clerocidin and dh-EPI stimulated cleavage whereas VM-26 was only weakly effective. Some clerocidin cleavage sites did not undergo spontaneous reversion, indicating that this agent can stimulate irreversible cleavage in vivo. Direct genomic sequencing showed that many clerocidin and dh-EPI sites, although distinct, mapped to the transcription start and to the proximal promoter of the H2A gene, suggesting that the region is highly accessible to topoisomerase II. Thus, the enzyme may play a role in maintaining a highly accessible chromatin structure under normal cell growth conditions, possibly mediated by specialised protein complexes. This study demonstrates that the use of distinct poisons greatly improves the definition of genomic sites of topoisomerase II activity.

A new Drosophila ultraviolet light-damaged DNA recognition endonuclease that selectively nicks a (6-4) photoproduct site

We have previously described the purification of an ultraviolet light (UV) damage-specific DNA-binding protein from Drosophila melanogaster, designated D-DDB P1 [Nucleic Acids Res., 23 (1995) 2600–2607]. Here, we obtained highly purified D-DDB P1 from Drosophila Kc cells, and we found that D-DDB P1 is also a nuclease. D-DDB P1 can selectively bind to pyrimidine (6-4) pyrimidone photoproducts, and in the presence of Mg ++, D-DDB P1 can catalyze an incision immediately on the 3′ and 5′ sides of the (6-4) photoproduct site.

In vivo association of lamins with nucleic acids in Drosophila melanogaster.

A 32P-labeling strategy was developed to study the interaction(s) in tissue culture cells between proteins and nucleic acids. Interphase and mitotic nuclear lamins were studied in Drosophila Kc cells. After bromodeoxyuridine incorporation and in vivo photo-crosslinking with 366 nm light, it was found that interphase lamins were associated with nucleic acid. Interactions with DNA as well as RNA were detected. In contrast, interaction of nucleic acids with mitotic lamin was not observed. Photo-crosslinking in the presence of antibiotics distamycin and/or chromomycin suggested that interphase lamins interacted with both A-T-rich DNA and G-C-rich DNA; interactions with G-C-rich DNA predominated. These results have implications for understanding the interphase organization of the higher eukaryotic cell nucleus as well as the transition of cells from interphase to mitosis. A model of nuclear organization, consistent with our results, is proposed.

Identification of CFDD (Common Regulatory Factor for DNA Replication and DREF Genes) and Role of Its Binding Site in Regulation of the Proliferating Cell Nuclear Antigen Gene Promoter

The Drosophila proliferating cell nuclear antigen (PCNA) gene promoter contains at least three transcriptional regulatory elements, the URE (upstream regulatory element), DRE (DNA replication-related element), and E2F recognition sites. In nuclear extracts of Drosophila Kc cells, we detected a novel protein factor(s), CFDD (common regulatory factor for DNA replication and DREF genes) that appeared to recognize two unique nucleotide sequences (5'-CGATA and 5'-CAATCA) and bind to three sites in the PCNA gene promoter. These sites were located at positions -84 to -77 (site 1), -100 to -93 (site 2) and -134 to -127 (site 3) with respect to the transcription initiation sites. Sites 2 and 3 overlapped with DRE and URE, respectively, and the 5'-CGATA matched with the reported recognition sequence of BEAF-32 (boundary element-associated factor of 32 kDa). Detailed analyses of CFDD recognition sequences and experiments with specific antibodies to DREF (DRE-binding factor) and BEAF-32 suggest that CFDD is different from DREF, UREF (URE-binding factor) and BEAF-32. A UV cross-linking experiment revealed that polypeptides of approximately 76 kDa in the nuclear extract interact directly with the CFDD site 1 sequence. Transient expression assays of chloramphenicol acetyltransferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying mutations in CFDD site 1 and examination of lacZ expression from PCNA promoter-lacZ fusion genes carrying mutations in site 1, introduced into flies by germ line transformation, revealed that CFDD site 1 plays an important role for the promoter activity both in cultured cells and in living flies. In addition to the PCNA gene, multiple CFDD sites were found in promoters of the DNA polymerase alpha and DREF genes, and CFDD binding to the DREF promoter was confirmed. Therefore, CFDD may play important roles in regulation of Drosophila DNA replication-related genes.

Identification of a Drosophila 60S ribosomal protein

We have previously reported the cloning and sequencing of a cDNA from Drosophila (DL43; GenBank Accession # U40226) which predicts a small protein homologous to a ribosomal protein in yeast and rat. In this study we show that a 330 nucleotide transcript encoding the putative ribosomal protein is expressed in Drosophila Kc cells under normal growth conditions (25°C). This :RNA is associated with the translational machinery and is present on polysomes. The DL43 cDNA was transcribed using T7 RNA polymerase and the resulting transcripts translated in a wheat germ extract. SDS/PAGE analysis of the in vitro transcribed and translated DL43 cDNA yields a small protein of approximately 9 kDa. Using two-dimensional gelelectrophoresis we have established that the radiolabeled DL43 protein comigrates with a small basic protein in the 60S ribosomal subunit. The electrophoretic mobilities of the in vitro and in vivo synthesized DL43 proteins are indistinguishable providing support for the identity of this protein.

A cDNA encodes the Drosophila homolog of yeast 60S ribosomal protein YL43.

We describe the nucleotide sequence of a cDNA clone isolated from Drosophila Kc cells which encodes an amino acid sequence homologous to a 60S ribosomal protein from yeast (YL43) and rat (p23). The DL43 cDNA is 320 nucleotides in length and predicts a protein of 76 amino acids and a calculated molecular mass of 8.9 kiloDaltons. Northern blot analysis demonstrates the presence of the DL43 transcript under both control (25 degrees C) and heat shock (37 degrees C) conditions. The Drosophila protein shares an 86% identity over the first 22 amino acids with the yeast YL43 protein and a 60% identity over the entire length of the partial sequence available for this protein.

Assembly of specific SR protein complexes on distinct regulatory elements of the Drosophila doublesex splicing enhancer.

The Drosophila doublesex female-specific splicing enhancer consists of two classes of regulatory elements, six 13-nucleotide repeat sequences, and a single purine-rich element (PRE). Here, we show that the Drosophila regulatory proteins Transformer (Tra) and Transformer 2 (Tra2) recruit different members of the SR family of splicing factors to the repeats and the PRE. The complexes formed on the repeats in HeLa cell extract consist of Tra, Tra2, and the SR protein 9G8. in Drosophila Kc cell extract, Tra and Tra2 recruit the SR protein RBP1 to the repeats. These proteins are arranged in a specific order on the repeats, with the SR protein at the 5' end of each repeat, and Tra2 at each 3' end. Although Tra did not cross-link strongly to the repeats, its presence was essential for the binding of Tra2 to the 3' end of the repeat. Individual SR proteins were also recruited to the PRE by Tra and Tra2, but in this case they were SF2/ASF and dSRp30 in HeLa and Drosophila cell extracts, respectively. The binding of Tra2, Tra, and the specific SR proteins to the repeats or the PRE was highly cooperative within each complex. Thus, Tra2, which contains a single RNA binding domain, can recognize distinct sequences in the repeats and the PRE in conjunction with specific SR proteins. These observations show that the protein composition of each complex is determined by the RNA recognition sequence and specific interactions between SR proteins and Tra and Tra2.

The chicken lysozyme gene 5' MAR and the Drosophila histone SAR are electroelutable from encapsulated and digested nuclei.

Cultured chicken cells were encapsulated in agarose microbeads, lysed in a near-physiological buffer and resulting encapsulated nuclei were digested with a restriction enzyme and electroeluted. After removal of approximately 97% of the chromatin, the nuclear lamina, residual nucleoli and an internal nuclear network remained. The majority of nascent RNA was also recovered in digested and electroeluted nuclei. Surprisingly, however, the chicken lysozyme gene 5' MAR was quantitatively electroeluted from digested nuclei of expressing and non-expressing cells, as well as the promoter region and the coding sequence. When encapsulated nuclei were digested partially, the proportion of elutable 5' MAR chromatin was comparable to that of elutable bulk chromatin. Furthermore, after digestion of encapsulated nuclei from Drosophila Kc cells, the histone SAR was electroeluted to the same extent as bulk chromatin. We conclude that the lysozyme gene 5' MAR and the histone SAR are not permanently attached to a nuclear matrix or scaffold.

8- O-Acetylharpagide is a nonsteroidal ecdysteroid agonist

We have identified a novel nonsteroidal ecdysteroid agonist. This compound was isolated from a methanol extract of Ajuga reptans L. (Lamiaceae) and the structure was identified by spectroscopic methods as 8- O-acetylharpagide. We have characterised this compound as an ecdysteroid agonist in a transactivation assay using β-galactosidase as the reporter gene regulated by ecdysteroid response elements. In this assay, 8- O-acetylharpagide has an EC 50 of 22 μM. The compound also competes with tritiated-ponasterone A for binding to the Drosophila ecdysteroid receptor. Finally, it induces differentiation of Drosophila Kc cells as would be expected of an ecdysteroid agonist. This iridoid glycoside is common to several plant species and may play a role in the natural defense mechanisms of plants.

Cell cycle parameters in Aedes albopictus mosquito cells.

We have analyzed cell cycle parameters for the Aedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10-12 h. The duration of mitosis (M) was < or = 1 h, and the DNA synthesis phase (S) required 6 h. Unlike Drosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2 h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.

Purification of an RNA Polymerase II Transcript Release Factor from Drosophila

Factor 2 was previously identified in Drosophila Kc cell nuclear extract (KcN) as an activity suppressing the appearance of long transcripts (Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1987) J. Biol. Chem. 262, 3244-3255). A 154-kDa protein with factor 2 activity was purified to apparent homogeneity from KcN. An immobilized template assay indicated that factor 2 caused the release of transcripts by RNA polymerase II in an ATP-dependent manner. Some early elongation complexes were resistant to factor 2 action but became sensitive after treatment with 1 M KCl. In the absence of factor 2, transcription complexes still exhibited a low degree of processivity suggesting that factor 2 was only partially responsible for abortive elongation.

Isolation and Characterization of cDNA for DREF, a Promoter-activating Factor for Drosophila DNA Replication-related Genes

DREF, a transcription regulatory factor which specifically binds to the promoter-activating element DRE (DNA replication-related element) of DNA replication-related genes, was purified to homogeneity from nuclear extracts of Drosophila Kc cells. cDNA for DREF was isolated with the reverse-transcriptase polymerase chain reaction method using primers synthesized on the basis of partial amino acid sequences and following screening of cDNA libraries. Deduced from the nucleotide sequences of cDNA, DREF is a polypeptide of 701 amino acid residues with a molecular weight of 80,096, which contains three characteristic regions, rich in basic amino acids, proline, and acidic amino acids, respectively. Deletion analysis of bacterially expressed DREF fused with glutathione S-transferase (GST-DREF) indicated that a part of the N-terminal basic amino acid region (16-115 amino acids) is responsible for the specific binding to DRE. A polyclonal and four monoclonal antibodies were raised against the GST-DREF fusion protein. The antibodies inhibited specifically the transcription of DNA polymerase alpha promoter in vitro. Cotransfection experiments using Kc cells demonstrated that overproduction of DREF protein overcomes the repression of the proliferating cell nuclear antigen gene promoter by the zerknüllt gene product. These results confirmed that DREF is a trans-activating factor for DNA replication-related genes. Immunocytochemical analysis demonstrated the presence of DREF polypeptide in nuclei after the eighth nuclear division cycle, suggesting that nuclear accumulation of DREF is important for the coordinate zygotic expression of DNA replication-related genes carrying DRE sequences.

A Novel Ecdysteroid Responsive Reporter Plasmid Regulated by the 5'-Upstream Region of the Drosophila melanogaster (Diptera: Drosophilidae) Acetylcholinesterase Gene

Acetylcholinesterase activity induction by ecdysteroids is known in Drosophila melanogaster Kc cells, but its induction at the gene expression level has not been studied. A reporter plasmid was constructed by ligation of the D. melanogaster acetylcholinesterase gene (Ace gene) 5'-upstream 1.62 kb region to firefly luciferase cDNA. The plasmid was then introduced into Kc cells by the electroporation method for induction study. With the addition of 1.0 × 10-8 M 20-hydroxyecdysone, a 4-fold induction of luciferase was observed 72 h after the hormone treatment, while 1.0 × 10-6 M ecdysone was required to give a 5-fold induction. Dibenzoyl hydrazines (RH 5849 and RH 5992), which are known as non-steroidal ecdysteroid agonists, also induced a 3 to 4-fold induction, whoever, RH 5992 showed a higher induction potential than RH 5849 (as in other ecdysteroid assays). These results suggest that this novel ecdysteroid responsive reporter plasmid (system) might be useful for the evaluation of ecdysteroids and further analysis of the acetylcholinesterase induction mechanism by ecdysteroids.

Localization of Sequences Required for Size-specific Splicing of a Small DrosophilaIntron in Vitro

Many introns in Drosophilaand other invertebrates are less than 80 nucleotides in length, too small to be recognized by the vertebrate splicing machinery. Comparison of nuclear splicing extracts from human HeLa and DrosophilaKc cells has revealed species-specificity, consistent with the observed size differences. Here we present additional results with the 68 nucleotide fifth intron of the Drosophilamyosin heavy chain gene. As observed with the 74 nucleotide second intron of the Drosophilawhite gene, the wild-type myosin intron is accurately spliced in a homologous extract, and increasing the size by 16 nucleotides both eliminates splicing in the Drosophilaextract and allows accurate splicing in the human extract. In contrast to previous results, however, an upstream cryptic 5′ splice site is activated when the wild-type myosin intron is tested in a human HeLa cell nuclear extract, resulting in the removal of 98 nucleotide intron. The size dependence of splicing in Drosophilaextracts is also intron-specific; we noted that a naturally larger (150 nucleotide) intron from the ftzgene is efficiently spliced in Kc cell extracts that do not splice enlarged introns (of 84, 90, 150 or 350 nucleotides) derived from the 74 nucleotide whiteintron. Here, we have exploited that observation, using a series of hybrid introns to show that a region of 46 nucleotides at the 3′ end of the whiteintron is sufficient to confer the species-specific size effect. At least two sequence elements within this region, yet distinct from previously described branchpoint and pyrimidine tract signals, are required for efficient splicing of small splicing of small hybrid introns in vitro.

Essential Role of E2F Recognition Sites in Regulation of the Proliferating Cell Nuclear Antigen Gene Promoter during Drosophila Development

We have found sequences similar to the transcription factor E2F recognition site within the Drosophila proliferating cell nuclear antigen (PCNA) gene promoter. These sequences are located at positions -43 to -36 (site I). and -56 to -49 (site II) with respect to the cap site Glutathione S-transferase (GST)-E2F and GST-DP fusion proteins cooperate and bind to the potential E2F sites in the PCNA promoter in vitro. A binding factor(s) to these sequences that has similar binding specificity to that of E2F was detected in nuclear extracts of Drosophila Kc cells. Furthermore, transient expression of target site for the activation coincided with the E2F sites. These results indicate that the PCNA gene is a likely target gene of E2F. Examination of lacZ expression from PCNA-lacZ fusion genes carrying mutations in either or both of two E2F sites introduced into flies by germ line transformation revealed that site II plays a major role in the PCNA promoter activity during embryogenesis and larval development, although both sites are required for optimal promoter activity. However, for maternal expression in ovaries, either one of the two sites is essentially sufficient to direct optimal promoter activity. These results demonstrate, for the first time, an essential role for E2F sites in regulation of PCNA promoter activity during development of a multicellular organism.

Purification of P-TEFb, a Transcription Factor Required for the Transition into Productive Elongation

Production of full-length runoff transcripts in vitro and functional mRNA in vivo is sensitive to the drug 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). We previously proposed the existence of an activity, P-TEF (positive transcription elongation factor) that functions in a DRB-sensitive manner to allow RNA polymerase II elongation complexes to efficiently synthesize long transcripts (Marshall, N. F. and Price, D. H. (1992) Mol. Cell. Biol. 12, 2078-2090). We have fractionated nuclear extracts of Drosophila melanogaster Kc cells and identified three activities, P-TEFa, factor 2, and P-TEFb, that are directly involved in reconstructing DRB-sensitive transcription. P-TEFb is essential for the production of DRB-sensitive long transcripts in vitro, while P-TEFa and factor 2 are stimulatory. P-TEFb activity is associated with a protein comprising two polypeptide subunits with apparent molecular masses of 124 and 43 kDa. Using a P-TEFb-dependent transcription system, we show that P-TEFb acts after initiation and is the limiting factor in the production of long run-off transcripts.

Distribution of PCNA during postblastoderm cell division cycles in the Drosophila melanogaster embryo: effect of a string- mutation.

We used immunocytochemical methods and a specific antibody to identify proliferating cell nuclear antigen (PCNA) in Drosophila embryos during the postblastoderm cell division cycles. The strong nuclear staining observed during interphase disappeared at prophase, staining remained nil throughout the remaining mitotic (M) phases and reappeared in nuclei at the next interphase. As the embryos with the homozygous string- mutation sustained the strong staining signal in nuclei throughout embryonic development, disappearance of the staining signal probably depends on string function and is coupled with the onset of mitosis. When cells in embryos were arrested at M phase following treatment with TN-16 or Vinblastine, the staining signal with the anti-PCNA antibody was lost. However, Western blots showed that the level of PCNA protein in M phase-arrested embryos did not decrease. Therefore, the disappearance of staining signals is apparently due to reorganization of PCNA protein in the multiprotein complex in nuclei, rendering it inaccessible to the antibody, rather than to the degradation of PCNA protein. In contrast to findings in developing embryos, cultured Drosophila Kc cells stained strongly with the anti-PCNA antibody, during both interphase and the M phase. In genetic crossing experiments of transgenic flies carrying the lacZ gene under the control of the PCNA gene regulatory region (-607 to +137 with respect to the transcription initiation site) with string- mutant files, the PCNA gene promoter seems to function in a manner independent of cell cycle progression or of functions of the string gene.