Chriz, a chromodomain protein specific for the interbands of Drosophila melanogaster polytene chromosomes.

Polytene interphase chromosomes are compacted into a series of bands and interbands reflecting their organization into independent chromosomal domains. In order to understand chromosomal organization, we set out to study the role of proteins that are selective for interbands. Here we describe the Drosophila melanogaster chromodomain protein Chriz that is coimmunoprecipitated with the zinc finger protein Z4. Both proteins colocalize exclusively to the interbands on Drosophila polytene chromosomes. Like Z4, Chriz is ubiquitously expressed throughout development and is associated with chromatin in all interphase nuclei. Following dissociation from chromatin, early in mitosis Chriz binds to the centrosomes and to the mitotic spindle. Newly induced amorphic Chriz alleles are early lethal, and ubiquitous overexpression of Chriz is lethal as well. Available Chriz hypomorphs which survive until pupal stage have a normal chromosomal phenotype. Reducing Z4 protein does not affect Chriz binding to polytene chromosomes and vice versa. Z4 is still chromosomally bound when Chriz protein is depleted by RNA interference.

[Construction of pMH, a convenient Escherichia coli protein expression vector]. / pMH--udobnyi vektor dlia produktsii belkov v Escherichia coli.

Here we describe the construction of a new vector, pMH, designed for protein expression in E. coli. The vector provides inducible and powerful T7 RNA polymerase driven transcription of the sequences introduced, and a polylinker comprising now 10 most widely used restriction sites, which allows virtually any sequence to be cloned. Cloning in-frame with the N-terminal (c-myc)3-(His)6-tag makes it possible, first, to easily affinity purify the proteins being expressed and, second, to detect the recombinant proteins with the antibodies specific for any of the tags when protein-specific antibodies are unavailable. General utility of pMH was demonstrated by successful expression in E. coli and further purification of Drosophila melanogaster Chriz (CG10712) product and of a number of its C-terminal truncations, with the approximate protein yeild constituting 10 mg/l culture.

[Use of immunogold labelling technique for immunoelectron microscope localization of proteins in Drosophila polytene chromosomes]. / Immunoélektronno-mikroskopicheskaia lokalizatsiia belkov na politennykh khromosomakh drozofily s pomoshch'iu immunnoi tekhniki mecheniia zolotom.

Using gold labeled antibodies, we developed and tested an immunoelectron microscope (IEM) method for detection of protein localization in Drosophila melanogaster polytene chromosomes. This method is based on procedures widely used for indirect immunofluorescent (IF) staining of salivary gland polytene chromosome squashes. The application of IEM was evaluated by using specific antibodies against proteins earlier localized in both decondensed (interbands and puffs) and compact (bands) regions of polytene chromosomes. In all the experiments, IEM and IF images for homologous chromosome regions were compared. When applied to regions of loose structures, IEM enabled us to localize, with high precision, signals in fine bands, interbands and puffs. There was a good correspondence between immunogold EM and IF data. However, there was no correspondence for dense bands: gold particles were distributed at their boundaries, while the entire bands showed bright fluorescence. This discrepancy probably resulted from a poor penetration of antibodies conjugated to gold particles in the tightly packaged structures. From the results obtained it may by concluded that the IEM method is advantageous for studying the fine protein topography of loose decompacted regions of polytene chromosomes. And this must be taken into consideration when protein localization in polytene chromosomes is performed.

Genetic and molecular complexity of the position effect variegation modifier mod(mdg4) in Drosophila.

mod(mdg4), also known as E(var)3-93D, is involved in a variety of processes, such as gene silencing in position effect variegation (PEV), the control of gypsy insulator sequences, regulation of homeotic gene expression, and programmed cell death. We have isolated a large number of mod(mdg4) cDNAs, representing 21 different isoforms generated by alternative splicing. The deduced proteins are characterized by a common N terminus of 402 amino acids, including the BTB/POZ-domain. Most of the variable C termini contain a new consensus sequence, including four positioned hydrophobic amino acids and a Cys(2)His(2) motif. Using specific antibodies for two protein isoforms, we demonstrate different distributions of the corresponding proteins on polytene chromosomes. Mutations in the genomic region encoding exons 1-4 show enhancement of PEV and homeotic transformation and affect viability and fertility. Homeotic and PEV phenotypes are enhanced by mutations in other trx-group genes. A transgene containing the common 5' region of mod(mdg4) that is present in all splice variants known so far partially rescues the recessive lethality of mod(mdg4) mutant alleles. Our data provide evidence that the molecular and genetic complexity of mod(mdg4) is caused by a large set of individual protein isoforms with specific functions in regulating the chromatin structure of different sets of genes throughout development.

Analysis of Drosophila salivary gland, epidermis and CNS development suggests an additional function of brinker in anterior-posterior cell fate specification.

Salivary glands are simple structured organs which can serve as a model system in the study of organogenesis. Following a large EMS mutagenesis we have identified a number of genes required for normal salivary gland development. Mutations in the locus small salivary glands-1 (ssg-1) lead to a drastic reduction in the size of the salivary glands. The gene ssg-1 was cloned and subsequent sequence and genetic analysis showed identity to the recently published gene brinker. The salivary gland placode in brinker mutants appears reduced along both the anterior-posterior and dorso-ventral axis. Analysis of the brinker cuticle phenotype revealed a similar loss of anterior-posterior as well as lateral cell fates. The abdominal ventral denticle belts show a reduced number of setae in the first denticle row. Furthermore, we observed a preferential loss of lateral neuroblasts in the anterior parasegment. Together, these phenotypes suggest that brinker not only plays a role in dorso-ventral but also in anterior-posterior axis patterning.

The protein Hrb57A of Drosophila melanogaster closely related to hnRNP K from vertebrates is present at sites active in transcription and coprecipitates with four RNA-binding proteins.

The hnRNP K protein is among the major hnRNA-binding proteins with a strong preference for cytidine-rich sequences. We have cloned a Drosophila hnRNP protein closely related to this vertebrate protein. The protein first identified by the monoclonal antibody Q18 is encoded by a gene located in 57A on polytene chromosomes and has been consequently named Hrb57A. The amino acid sequence of the Hrb57A KH domains and their overall organisation in the protein are remarkably similar to the vertebrate proteins. As the hnRNP K in vertebrates the M(r) 55 000 Drosophila Hrb57A/Q18 protein strongly binds to poly(C) in vitro and is ubiquitously present in nuclei active in transcription. On polytene chromosomes it is found in many puffs and minipuffs. Hrb57A/Q18 specifically coprecipitates four other proteins: Hrb87F/P11 a Drosophila hnRNP A1 homologue, the hnRNA-binding protein S5, the RNA recognition motif-containing protein NonA and the RNA-binding zinc finger-containing protein on ecdysone puffs PEP/X4.

X-linked loci of Drosophila melanogaster causing defects in the morphology of the embryonic salivary glands.

To identify X chromosomal genes required for salivary gland development in the Drosophila embryo, we screened embryos hemizygous for EMS-induced lethal mutations to find mutations causing gross morphological defects in salivary gland development. The parental strain carried a lac Z transgene on the second chromosome, which was specifically expressed in the salivary glands so the mutations could be unambiguously identified. Embryos from 3,383 lines were tested for salivary gland abnormalities following lacZ staining. From 63 lines exhibiting aberrant salivary gland phenotypes, 52 stable lines were established containing mutations affecting salivary gland development. From these, 39 lines could be assigned to nine complementation groups: armadillo, brinker, folded gastrulation, giant, hindsight, Notch, runt, stardust and twisted gastrulation.

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.

The puff-specific RRM protein NonA is a single-stranded nucleic acid binding protein.

The chromatin protein NonA from Drosophila, present in many puffs on polytene chromosomes, belongs to the growing class of RRM proteins. Exchange of amino acids within the RNP1 and RNP2 consensus sequences, known from other RRM proteins to be essential for RNA binding, has been shown drastically to reduce NonA function in vivo. Here we compare NonA binding to RNA from the Sgs-4 gene, an in situ target for NonA, with binding to Sgs-3 RNA, which is not a target of NonA. Using an immunoprecipitation assay in vitro we show that NonA binds to single-stranded (ss)DNA and RNA with moderate affinity (KD=8x10(-8) M). However, we did not observe sequence-specific binding to the Sgs-4 transcript nor to Sgs-4 DNA containing upstream regulatory sequences. Point mutations within the RNP1 and RNP2 consensus sequences that interfere with NonA function in vivo do not significantly change chromosomal binding nor the general affinity for RNA. The expression of Sgs-4 RNA relative to the expression of Sgs-3 RNA remains the same in the presence or absence of NonA protein. com/link/service/journals/00412/bibs/108n3p162.html

Molecular screening for P-element insertions in a large genomic region of Drosophila melanogaster using polymerase chain reaction mediated by the vectorette.

As an alternative to existing methods for the detection of new insertions during a transposon mutagenesis, we adapted the method of vectorette ligation to genomic restriction fragments followed by PCR to obtain genomic sequences flanking the transposon. By combining flies containing a defined genomic transposon with an excess of flies containing unrelated insertion sites, we demonstrate the specificity and sensitivity of the procedure in the detection of integration events. This method was applied in a transposon-tagging screen for BJ1, the Drosophila homolog of the vertebrate gene Regulator of Chromosome Condensation (RCCI). Genetic mobilization of a single genomic P element was used to generate preferentially new local insertions from which integrations into a genomic region surrounding the BJ1 gene were screened. Flies harboring new insertions were phenotypically selected on the basis of the zeste1-dependent transvection of white. We detected a single transposition to a 13-kb region close to the BJ1 gene among 6650 progeny that were analyzed. Southern analysis of the homozygous line confirmed the integration 3 kb downstream of BJ1.

The dynamic nuclear redistribution of an hnRNP K-homologous protein during Drosophila embryo development and heat shock. Flexibility of transcription sites in vivo.

The Drosophila protein Hrb57A has sequence homology to mammalian heterogenous nuclear ribonucleoprotein (hnRNP) K proteins. Its in vivo distribution has been studied at high resolution by confocal laser scanning microscopy (CLSM) in embryos injected with fluorescently labeled monoclonal antibody. Injection of antibody into living embryos had no apparent deleterious effects on further development. Furthermore, the antibody-protein complex could be observed for more than 7 cell cycles in vivo, revealing a dynamic redistribution from the nucleus to cytoplasm at each mitosis from blastoderm until hatching. The evaluation of two- and three-dimensional CLSM data sets demonstrated important differences in the localization of the protein in the nuclei of living compared to fixed embryos. The Hrb57A protein was recruited to the 93D locus upon heat shock and thus serves as an in vivo probe for the activity of the gene in diploid cells of the embryo. Observations during heat shock revealed considerable mobility within interphase nuclei of this transcription site. Furthermore, the reinitiation as well as the down regulation of transcriptional loci in vivo during the recovery from heat shock could be followed by the rapid redistribution of the hnRNP K during stress recovery. These data are incompatible with a model of the interphase nucleus in which transcription complexes are associated with a rigid nuclear matrix.

Bioassaying putative RNA-binding motifs in a protein encoded by a gene that influences courtship and visually mediated behavior in Drosophila: in vitro mutagenesis of nonA.

The no-on-transient-A (nonA) gene of Drosophila melanogaster influences vision, courtship song, and viability. The nonA-encoded polypeptide is inferred to bind single-stranded nucleic acids. Although sequence-analysis of NONA implies that it belongs to a special interspecific family of this protein type, it does contain two classical RNA recognition motifs (RRM). Their behavioral significance was assayed by generating transgenic strains that were singly or multiply mutated within the relatively N-terminal motif (RRM1) or within RRM2. Neither class of mutation affected NONA binding to polytene chromosomes. The former mutations led to extremely low viability, accompanied by diminished adult longevities that were much worse than for a nonA-null mutant, implying that faulty interpolypeptide interactions might accompany the effects of the amino-acid substitutions within RRM1. All in vitro-mutated types caused optomotor blindness and an absence of transient spikes in the electroretinogram. Courtship analysis discriminated between the effects of the mutations: the RRM2-mutated type generated song pulses and trains that tended to be mildly mutant. These phenotypic abnormalities reinforce the notion that nonA's ubiquitous expression has its most important consequences in the optic lobes, the thoracic ganglia, or both, depending in part on the nonA allele.

Time-resolved, in vivo studies of mitotic spindle formation and nuclear lamina breakdown in Drosophila early embryos.

Time-resolved, two-component, three-dimensional fluorescence light microscopy imaging in living Drosophila early embryos is used to demonstrate that a large fraction of the nuclear envelope lamins remain localized to a rim in the nuclear periphery until well into metaphase. The process of lamin delocalization and dispersal, typical of 'open' forms of mitosis, does not begin until about the time the final, metaphase geometry of the mitotic spindle is attained. Lamin dispersal is completed about the time that the chromosomal movements of anaphase begin. This pattern of nuclear lamina breakdown appears to be intermediate between traditional designations of 'open' and 'closed' mitoses. These results thus clarify earlier observations of lamins in mitosis in fixed Drosophila early embryos, clearly showing that the observed lamin localization does not result from a structurally defined 'spindle envelope' that persists throughout mitosis. During this extended time interval of lamin localization in the nuclear periphery, the lamina undergoes an extensive series of structural rearrangements that are closely coupled to, and likely driven by, the movements of the centrosomes and microtubules that produce the mitotic spindle. Furthermore, throughout this time the nuclear envelope structure is permeable to large macromolecules, which are excluded in interphase. While the functional significance of these structural dynamics is not yet clear, it is consistent with a functional role for the lamina in mitotic spindle formation.

The enhancer of position-effect variegation of Drosophila, E(var)3-93D, codes for a chromatin protein containing a conserved domain common to several transcriptional regulators.

In Drosophila modifying mutations of position-effect variegation have been successfully used to genetically dissect chromatin components. The enhancer of position-effect variegation E(var)3-93D [formerly E-var(3)3] encodes proteins containing a domain common to the transcriptional regulators tramtrack and the products of the Broad complex. It interacts with a number of chromatin genes that suppress position-effect variegation. Mutations in E(var)3-93D exhibit an imprinting-like effect on the Y chromosome. This effect is transmitted paternally over several generations. Homeotic transformations in E(var)3-93D mutants indicate an involvement of the gene products in regulation of homeotic gene complexes. An antiserum raised against E(var)3-93D protein detects this chromosomal protein in a large subset of sites in polytene chromosomes. Our genetic and molecular data suggest that the proteins of E(var)3-93D are generally involved in establishing and/or maintaining an open chromatin conformation.

Genetic and molecular analysis of the X chromosomal region 14B17-14C4 in Drosophila melanogaster: loss of function in NONA, a nuclear protein common to many cell types, results in specific physiological and behavioral defects.

We have performed a genetic analysis of the 14C region of the X chromosome of Drosophila melanogaster to isolate loss of function alleles of no-on-transient A (nonA; 14C1-2; 1-52.3). NONA is a nuclear protein common to many cell types, which is present in many puffs on polytene chromosomes. Sequence data suggest that the protein contains a pair of RNA binding motifs (RRM) found in many single-strand nucleic acid binding proteins. Hypomorphic alleles of this gene, which lead to aberrant visual and courtship song behavior, still contain normally distributed nonA RNA and NONA protein in embryos, and in all available alleles NONA protein is present in puffs of third instar larval polytene chromosomes. We find that complete loss of this general nuclear protein is semilethal in hemizygous males and homozygous cell lethal in the female germline. Surviving males show more extreme defects in nervous system function than have been described for the hypomorphic alleles. Five other essential genes that reside within this region have been partially characterized.

In vivo observation of the puff-specific protein no-on transient A (NONA) in nuclei of Drosophila embryos.

The spatial distribution of no-on transient A (NONA), a protein associated with specific puffs on polytene chromosomes, was followed in nuclei of living Drosophila embryos by microinjection of fluorescently labeled monoclonal antibody to NONA. The injected antibodies remained active until the larval stage, revealing the distribution of the NONA protein throughout embryogenesis. Most injected animals completed embryonic development and hatched as normal larvae. NONA was restricted to the cytoplasm until the end of cycle 11. We document an active uptake of the NONA-antibody complex into early interphase nuclei from nuclear cycle 14 onwards, following each mitosis. Significant differences in the distribution of the protein between fixed and living embryos were apparent, particularly at high resolution. The NONA protein was localized in the nuclei of living embryos at discrete sites, most of which lay at the periphery and some of which were tightly clustered. The constellation of sites changed with time; in some nuclei these changes were fast whereas in other nuclei the pattern was quite stable. These data suggest that specific protein complexes associated with active interphase chromatin, and possibly chromatin in general, are mobile in the living organism.

Consequences of topoisomerase II inhibition in early embryogenesis of Drosophila revealed by in vivo confocal laser scanning microscopy.

The regulation of DNA topology by topoisomerase II from Drosophila melanogaster has been studied extensively by biochemical methods but little is known about its roles in vivo. We have performed experiments on the inhibition of topoisomerase II in living Drosophila blastoderm embryos. We show that the enzymatic activity can be specifically disrupted by microinjection of antitopoisomerase II antibodies as well as the epipodophyllotoxin VM26, a known inhibitor of topoisomerase II in vitro. By labeling the chromatin of live embryos with tetramethylrhodamine-coupled histones, the effects of inhibition on nuclear morphology and behaviour was followed in vivo using confocal laser scanning microscopy. Both the antibodies and the drug prevented or hindered the segregation of chromatin daughter sets at the anaphase stage of mitosis. In addition, high concentrations of inhibitor interfered with the condensation of chromatin and its proper arrangement into the metaphase plate. The observed effects yielded non-functional nuclei, which were drawn into the inner yolk mass of the embryo. Concurrently, undamaged nuclei surrounding the affected region underwent compensatory division, leading to the restoration of the nuclear population, and thereby demonstrating the regulative capacity of Drosophila blastoderm embryos.

Identification of an essential Drosophila gene that is homologous to the translation initiation factor eIF-4A of yeast and mouse.

A gene encoding a protein homologous to the translation initiation factor eIF-4A in mouse has been identified in Drosophila melanogaster. The predicted amino acid sequence shows 73% identity with the mouse gene and 67% identity with a homologous protein from yeast. The single-copy Drosophila gene is located on chromosome arm 2L at 26A7-9. Several recessive lethal mutations have been isolated and genetically characterized. Northern blot hybridization shows two abundant transcripts of 1.75 kb and 1.9 kb throughout all developmental stages. Both transcripts are maternally provided to the oocyte.

The Drosophila nuclear protein Bx42, which is found in many puffs on polytene chromosomes, is highly charged.

The Drosophila nuclear protein Bx42 is present in a set of transcriptionally active puffs on polytene chromosomes. cDNA clones coding for this protein were isolated from a lambda gt11 expression library. The two Bx42 transcripts are ubiquitously expressed and are already detectable in early stages of development. The corresponding genomic region, in 8C7-8, was isolated and sequenced. Both transcripts direct the production of the same basic, highly charged 547 amino acid protein with a calculated 61.1 kDa molecular weight.