Transcriptional regulation of the Drosophila gene zen by competing Smad and Brinker inputs.

The establishment of expression domains of developmentally regulated genes depends on cues provided by different concentrations of transcriptional activators and repressors. Here we analyze the regulation of the Drosophila gene zen, which is a target of the Decapentaplegic (Dpp) signaling pathway during cellular blastoderm formation. We show that low levels of the Dpp signal transducer p-Mad (phosphorylated Mad), together with the recently discovered negative regulator Brinker (Brk), define the spatial limits of zen transcription in a broad dorsal-on/ventral-off domain. The subsequent refinement of this pattern to the dorsal-most cells, however, correlates with high levels of p-Mad that accumulate in the same region during late blastoderm. Examination of the zen regulatory sequences revealed the presence of multiple Mad and Brk binding sites, and our results indicate that a full occupancy of the Mad sites due to high concentrations of nuclear Mad is the primary mechanism for refinement of zen. Interestingly, several Mad and Brk binding sites overlap, and we show that Mad and Brk cannot bind simultaneously to such sites. We propose a model whereby competition between Mad and Brk determines spatially restricted domains of expression of Dpp target genes.

The Drosophila gene brinker reveals a novel mechanism of Dpp target gene regulation.

decapentaplegic (dpp), a Drosophila member of the TGFbeta family of secreted molecules, functions as a long-range morphogen in patterning of the embryo and the adult appendages. Dpp signals via the SMAD proteins Mad and Medea. Here we show that in the absence of brinker (brk), Mad is not required for the activation of Dpp target genes that depend on low levels of Dpp. brk encodes a novel protein with features of a transcriptional repressor. brk itself is negatively regulated by Dpp. Dpp signaling might relieve brk's repression of low-level target genes either by transcriptional repression of brk or by antagonizing a repressor function of brk at the target gene promoters.

Isolation and characterization of a new gene encoding a member of the HIRA family of proteins from Drosophila melanogaster.

The HIRA family of genes (named after yeast HIR genes; HIR is an acronym for 'histone regulator') includes the yeast HIR1 and HIR2 repressors of histone gene transcription in S. cerevisiae, human TUPLE-1/HIRA, chicken HIRA, and mouse HIRA. Here, we describe a new member of the HIRA family, Dhh, for the Drosophila homolog of HIRA . Northern analysis with poly (A)+ mRNA isolated from different developmental stages of Drosophila melanogaster shows hybridization with a single Dhh transcript of 4.1kb. Hybridization is strong in female adults, unfertilized eggs and 0-3-h-old embryos, then diminishes, but is still detectable, during later stages of development and in adult males. More specifically, in-situ hybridization shows that Dhh transcripts, which are initially detected in nurse cells during mid-oogenesis, become localized to the developing oocyte at high levels. Transcripts persist strongly during early blastoderm stages then fade dramatically by 3h of development. The Dhh cDNA encodes an open reading frame of 1061 amino acids with high similarity scores to the HIRA polypeptides, as well as two hypothetical polypeptides from C. elegans and S. pombe, in a protein database search. They all share three highly homologous regions: a WD-repeat cluster, a small domain with clustered positively charged amino acids, and a domain comprising two repeats with close resemblance to WD repeats plus a region with no homology outside of the family. The conservation of these homologous regions in HIRA-encoded proteins from evolutionary distant organisms suggests that they are important for the activity of the members of the family.

puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila.

The activation of MAPKs is controlled by the balance between MAPK kinase and MAPK phosphatase activities. The latter is mediated by a subset of phosphatases with dual specificity (VH-1 family). Here, we describe a new member of this family encoded by the puckered gene of Drosophila. Mutations in this gene lead to cytoskeletal defects that result in a failure in dorsal closure related to those associated with mutations in basket, the Drosophila JNK homolog. We show that puckered mutations result in the hyperactivation of DJNK, and that overexpression of puc mimics basket mutant phenotypes. We also show that puckered expression is itself a consequence of the activity of the JNK pathway and that during dorsal closure, JNK signaling has a dual role: to activate an effector, encoded by decapentaplegic, and an element of negative feedback regulation encoded by puckered.

The transcriptional corepressor DSP1 inhibits activated transcription by disrupting TFIIA-TBP complex formation.

Transcriptional repression of eukaryotic genes is essential for many cellular and developmental processes, yet the precise mechanisms of repression remain poorly understood. The Dorsal Switch Protein (DSP1) was identified in a genetic screen for activities which convert Dorsal into a transcriptional repressor. DSP1 shares structural homology with the HMG-1/2 family and inhibits activation by the rel transcription factors Dorsal and NF-kappaB in transfection studies. Here we investigate the mechanism of transcriptional repression by DSP1. We found that DSP1 protein can act as a potent transcriptional repressor for multiple activator families in vitro and in transfection studies. DSP1 bound directly to the TATA binding protein (TBP), and formed a stable ternary complex with TBP bound to DNA. DSP1 preferentially disrupted the DNA binding of TBP complexes containing TFIIA and displaced TFIIA from binding to TBP. Consistent with the inhibition of TFIIA-bound complexes, DSP1 was shown to inhibit activated but not basal transcription reactions in vitro. The ability of DSP1 to interact with TBP and to repress transcription was mapped to the carboxy-terminal domain which contains two HMG boxes. Our results support the model that DSP1 represses activated transcription by interfering with the binding of TFIIA, a general transcription factor implicated in activated transcription pathways.

The Drosophila dorsal morphogen represses the tolloid gene by interacting with a silencer element.

The dorsal protein (DL) regulates the transcriptional activity of several genes that determine cell fate along the dorsoventral axis of the Drosophila melanogaster embryo. DL is present at high levels in ventral nuclei, where it activates some genes (twi and sna) and represses others (zen, dpp, and tld). DL shows homology to the Rel family of proteins and interacts with specific DNA sequences in the regulatory regions of its target genes. The distal portion of the zen gene acts as a silencer that can mediate the repression of a heterologous promoter in ventral regions of the embryo. It contains four DL binding sites which alone are sufficient for activation but not repression. Here we analyze the interaction of DL with another one of its repressed targets, the tolloid (tld) gene. Approximately 800 bp of 5'-flanking sequences upstream of the tld coding region were shown to drive an expression pattern indistinguishable from the wild-type pattern. A 423-bp fragment located within these sequences contains two DL binding sites and was shown to act as a silencer to mediate ventral repression. Point mutations in the sites abolish not only DNA binding but also ventral repression. We discuss a comparison of the DNA sequences from the zen and tld promoters and the possible mechanisms of transcriptional silencing.

Conversion of a silencer into an enhancer: evidence for a co-repressor in dorsal-mediated repression in Drosophila.

The dorsal (dl) protein gradient determines patterns of gene expression along the dorsal-ventral axis of the Drosophila embryo. dl protein is at peak levels in ventral nuclei of the embryo where it activates some genes (twist and snail) and represses others [zerknullt (zen), decapentaplegic and tolloid]. It is a member of the rel family of transcription factors and interacts with specific DNA sequences in the regulatory regions of its target genes. These sequences (dl binding sites), when taken from the context of either an activated or repressed promoter, mediate transcriptional activation of a heterologous promoter, but not repression. We found that T-rich sequences close to the dl binding sites in the silencer region of the zen promoter are conserved between three Drosophila species. Using this sequence information we defined a minimal element that can mediate repression of a heterologous promoter. This element interacts with at least two factors present in embryonic extracts, one of which is dl protein. The other factor binds to the T-rich site. Point mutations in either site abolish ventral repression in vivo. In addition, mutations in the T-rich site cause ectopic expression in ventral regions indicating that the minimal silencer was converted into an enhancer.

In vitro transcription through nucleosomes by T7 RNA polymerase.

The present work examines the fate of nucleosomes after in vitro transcription of a 1400 bp DNA template containing the mouse alpha-globin sequences and the promoter of T7 RNA polymerase. Naked and nucleosome-bearing templates (containing about four or seven histone H1-lacking particles per template) have been studied by sedimentation, gel electrophoresis, digestion with restriction nucleases and electron microscopy. Both naked and nucleosome-organized templates could be transcribed in vitro by the T7 polymerase. With all types of templates, both full length and shorter transcripts were obtained. The incomplete transcripts were represented by many distinct bands, pointing to the presence of multiple stops in the process of elongation. The electrophoretic pattern of the transcripts was identical in naked and in nucleosome-containing templates, showing that the stops depended on some particular DNA sequences and not on the presence of nucleosomes. The efficiency of transcription in the presence of nucleosomes was decreased owing to three different factors: (i) blocked initiation in a fraction of the templates which had their promoters occupied by a nucleosome; (ii) a decreased rate of elongation and (iii) a lag period of initiation. Sedimentation velocity, electrophoretic mobility and protection of four different restriction sites of the templates demonstrated that T7 polymerase transcribed through nucleosomes without their displacement.

The complexity of 75S premessenger RNA in balbiani ring granules studied by a new RNA band retardation assay.

Under normal growth conditions, Balbiani ring granules constitute premessenger ribonucleoprotein (RNP) particles synthesized in two chromosomal puffs, Balbiani ring (BR) 1 and 2, in the larval salivary glands of Chironomus tentans. At least three genes encoding 75S RNA are present in these two BRs: one in BR1 and two in BR2 (BR2.1 and BR2.2). The complexity of BR granule 75S RNA was studied by agarose gel electrophoresis under non-denaturing conditions. We recorded three main bands, designated I, II and III. Experiments with denaturing gels demonstrated that the differences in migration reflected mainly, but not exclusively, conformational differences. Northern blotting experiments showed that band I contained BR1 sequences, band II contained BR2.1 sequences, and band III contained BR2.2 sequences. To study whether additional genes contributed to the BR granule 75S RNA, an RNA band shift assay was developed. When an oligodeoxyribonucleotide complementary to repetitive BR1 and BR2.2 sequences was hybridized to 75S RNA prior to electrophoresis, bands I and III were retarded but not band II. An oligonucleotide complementary to a repetitive BR2.1 sequence only shifted band II. Since no detectable 75S RNA remained unchanged in these experiments, and all bands were identified by Northern blotting, all the BR granules are likely to originate from the BR1, BR2.1 and BR2.2 genes; no additional genes have to be invoked. Possible applications of the new RNA band shift assay are discussed.

Segregation of nucleosomes in replicated mouse alpha-globin gene.

DNA of mouse erythroleukemia cells grown in vitro was labeled with bromodeoxyuridine during cycloheximide-inhibited protein synthesis. Isolated nuclei were digested with micrococcal nuclease to obtain monosomes and monosomal dsDNA. The protection of the heavy and of the light strands of the newly replicated DNA was studied by dot hybridization with the coding and with its complementary noncoding strand of the alpha-globin gene. The results show that both sides of the replication fork contain protected sequences of the gene, thus supporting a bilateral (dispersive) mode of nucleosome segregation during DNA replication.

Activated murine alpha-globin gene is not preferentially associated with the nuclear matrix.

The association of the murine alpha-globin gene with the nuclear matrix was studied in three different states of the gene: inactive (EAT cells), potentially active (MEL cells) and active (induced MEL cells). When "native" nuclei were digested with DNase I it was found that the nuclear matrix was not enriched in alpha-globin DNA sequences in all three different types of cells. A nuclease-hypersensitive site in the 5'-flanking region of the alpha-globin gene was detected in the induced MEL-cells.

[Unsaturated fatty acids in the lipids of the spermatozoa of Ovis amon aries L. and Sus scrofa domestica L]. / Nenasiteni mastni kiselini v lipidi ot spermatozoidi ot Ovis amon aries L. i Sus scrofa domestica L.

Spectrophotometric investigations were carried out with five unsaturated fatty acids found in the composition of spermatozoid lipids of animals belonging to four breeds of Ovis amon aries L. Parallel comparative analysis were made of the fatty acids in the lipids extracted from spermatozoa of Landrace swine of the Sus scrofa domestica L. species. Differences were established as to the percent content of fatty acids, having low points of freezing. It is believed that the criterion of cold resistance of the spermatozoid membrane lipids ranges within definite limits, depending on the maximum content of fatty acids with possibly lowest temperatures of freezing. The comparative investigations of both species of animals with regard to the percent ratio of spermatozoid fatty acids of low points of freezing have yielded results that speak in favour of the Ovis amon aries L. species. The different points of freezing below the level of the physiologically admissible temperature is referred to as one of the reasons for disturbances of the barrier functions of the membrane at deep freezing.

Presence of nucleosomal repeat in the transcribed alpha globin gene of induced murine erythroleukemia cells.

The sensitivity of the mouse alpha-globin gene to micrococcal nuclease and its nucleosomal repeat were studied in three different functional states of the gene: inactive in EAT cells, potentially active in uninduced MEL cells and active in induced MEL cells. The results show that: 1. The nuclease sensitivity of the gene differs in the three different functional states. 2. Both the coding and the 5'-flanking regions of the induced actively transcribed gene show a typical nucleosomal repeat pattern. 3. Hypersensitive sites for micrococcal nuclease and for an endogenous nuclease appear upstream of the gene after induction of differentiation.

Nuclear matrix and transcriptional activity of the mouse alpha-globin gene.

The association of the mouse alpha-globin gene with the nuclear matrix was studied when the gene was permanently repressed in Ehrlich ascites cells, potentially active in uninduced Friend cells or actively transcribed in induced Friend cells. Matrix-associated DNA was obtained by two methods, differing in the order of treatment of the nuclei with high salt and DNase I. By using a cloned alpha-globin probe, no enrichment in alpha-globin sequences was found in the matrix-associated DNA after DNase I digestion of high-salt treated nuclei from Ehrlich ascites and uninduced Friend cells. In induced Friend cells, a high enrichment (up to 20 times) of alpha-globin sequences was detected in the DNA left with the nuclear matrix structures. The size of the DNA fragments obtained by this procedure indicated a random attack and did not correspond to a progressive top-to-bottom cleavage model. No enrichment in alpha-globin sequences was found in induced Friend cells if nuclear matrices were obtained by DNase I digestion of the nuclei before the treatment with high salt. Our data suggest that the enrichment in actively transcribed genes of matrices from nuclei treated with high salt does not reflect a localization of these genes close to the attachment sites of the chromatin loops but rather their artefactual association with some high salt-insoluble proteins of the transcriptional complexes.