rolling pebbles (rols) is required in Drosophila muscle precursors for recruitment of myoblasts for fusion.

Mutations in the rolling pebbles (rols) gene result in severe defects in myoblast fusion. Muscle precursor cells are correctly determined, but myogenesis does not progress significantly beyond this point because recognition and/or cell adhesion between muscle precursor cells and fusion-competent myoblasts is disturbed. Molecular analysis of the rols genomic region reveals two variant transcripts of rols due to different transcription initiation sites, rols6 and rols7. rols6 mRNA is detectable mainly in the endoderm during differentiation as well as in malpighian tubules and in the epidermis. By contrast, rols7 expression is restricted to the mesoderm and later to progenitor descendants during somatic and pharyngeal muscle development. Transcription starts at the extended germ band stage when progenitor/founder cells are determined and persists until stage 13. The proteins encoded by the rols gene are 1670 (Rols6) and 1900 (Rols7) amino acids in length. Both forms contain an N-terminal RING-finger motif, nine ankyrin repeats and a TPR repeat eventually overlaid by a coiled-coil domain. The longer protein, Rols7, is characterized by 309 unique N-terminal amino acids, while Rols6 is distinguishable by 79 N-terminal amino acids. Expression of rols7 in muscle founder cells indicates a function of Rols7 in these cells. Transplantation assays of rols mutant mesodermal cells into wild-type embryos show that Rols is required in muscle precursor cells and is essential to recruit fusion-competent myoblasts for myotube formation.

Expression of the beta3 tubulin gene (beta Tub60D) in the visceral mesoderm of Drosophila is dependent on a complex enhancer that binds Tinman and UBX.

The beta3 tubulin gene of Drosophila is expressed in the major mesodermal derivatives during their differentiation. The gene is subject to complex stage- and tissue-specific transcriptional control by upstream as well as downstream regions. Analysis of the vm1 enhancer, which is responsible for tissue-specific expression in the visceral mesoderm and is localized in the intron, revealed a complex modular arrangement of regulatory elements. In vitro and in vivo experiments uncovered two binding sites, termed UBX1 and UBX2, for the product of the homeotic gene Ultrabithorax (Ubx), which are required for high-level expression in pPS6 and PS7. Further analysis of the vm1 enhancer revealed that deletion of a specific element, termed element 7 (e7), abolishes transcription of the lacZ reporter gene in all parasegments except pPS6/PS7. Gel-retardation and footprint analysis identified a binding site for the homeodomain protein Tinman, which is essential for the specification of the dorsal mesoderm, within e7. Simultaneous deletion of two further sequence blocks in the vml enhancer, named elements 3 (e3), and 6 (e6), results in a reduction analogous to that caused by removal of e7. The e6 sequence contains conserved motifs also found in the visceral enhancer of the Ubx gene. Therefore we conclude that these elements act in concert with the Tinman binding site to achieve high expression levels. Thus the vm1 enhancer of the beta3 tubulin gene contains a complex array of elements that are involved in transactivation by a combination of tissue- and position-specific factors including Tinman and UBX.

Independent regulatory elements in the upstream region of the Drosophila beta 3 tubulin gene (beta Tub60D) guide expression in the dorsal vessel and the somatic muscles.

The beta 3 tubulin gene (beta Tub60D) is a structural gene expressed during mesoderm development from the extended germ band stage onward. Expression within the individual mesodermal derivatives is guided by different control elements. The upstream regions allow expression in the dorsal vessel and the somatic mesoderm while enhancers localized in the first intron guide expression in the visceral mesoderm. Deletion analysis carried out in transgenic flies revealed independent regulatory elements for the dorsal vessel and the somatic mesoderm. For expression in the somatic mesoderm, a 279-bp region is absolutely essential. This region contains a binding site for the Drosophila myocyte-specific enhancer binding factor 2 (D-MEF2), a MADS-box transcription factor known to be essential for mesoderm development. Deletion or mutation of this D-MEF2 binding site strongly reduces transcription. This pattern is consistent with the strongly reduced expression of beta 3 tubulin in D-mef2 mutant embryos. This analysis furthermore reveals that the D-MEF2 binding site acts in concert with nearby cis regulatory elements. These data show that the upstream control region of the beta 3 tubulin gene is an early target of the D-MEF2 transcriptional activator.

Transcription of the beta 1 tubulin (beta Tub56D) gene in apodemes is strictly dependent on muscle insertion during embryogenesis in Drosophila melanogaster.

The insertion of the somatic musculature into the epidermis during embryogenesis of Drosophila melanogaster represents an excellent system for the investigation of cell-cell communication processes. Evidence from earlier experiments suggested that the expression of the beta 1 tubulin gene from D. melanogaster in the epidermal attachments may be dependent upon myotube insertion. Analysis of the transcription of the beta 1 tubulin gene in mutants for pair-rule or segment polarity genes revealed strong coupling of the induction process to myotube insertion. Involvement of extracellular matrix proteins of the integrin family could be excluded since beta 1 mRNA is detectable in the apodemes in mutants for the common subunit PS beta until muscles detach late in embryogenesis. Furthermore, the lack of specific muscles in the myogenic mutants rolling stone (rost) and not enough muscles (nem) eliminates beta 1 transcription exclusively in the corresponding apodemes. The data presented clearly show that the transcription of the beta 1 tubulin gene in the muscle attachment sites is activated by myotube insertion by a yet unidentified pathway.

Related enhancers in the intron of the beta1 tubulin gene of Drosophila melanogaster are essential for maternal and CNS-specific expression during embryogenesis.

Expression of the beta1 tubulin gene of Drosophila melanogaster is under complex developmental control. For high levels of transcription in the embryonic central nervous system (CNS) different modules dispersed over 3 kb have to co-operate. Combination of a core promoter with either far upstream localized enhancer elements or, alternatively, with an enhancer from the intron results in expression limited to only a few neuronal cells. Cooperation of all three modules, however, leads to high level expression in most neuronal cells of the CNS. In the intron, we identified a 6 bp core element which is essential for transcription in the CNS, as well as an 8 bp element required for maternal expression. Interestingly, both motifs are quite similar, with CAAAAT as the CNS core and CAAAAAT as the maternal enhancer core. Specific binding of proteins from nuclear extracts to the CNS-specific element could be demonstrated. We suggest that the beta1 tubulin gene represents an ideal marker gene to elucidate connections between pro-neural or neurogenic genes and downstream target genes throughout the CNS.

Muscle development and attachment to the epidermis is accompanied by expression of beta 3 and beta 1 tubulin isotypes, respectively.

In Drosophila beta tubulins are encoded by a small gene family whose members are differentially expressed in a highly cell and tissue specific manner. Here we focus on the expression of the beta 3 tubulin isotype during mesoderm differentiation and beta 1 tubulin expression in the apodemes during embryonic development. The beta 3 tubulin isotype is first detectable at the extended germband stage shortly before the separation of somatic and visceral derivatives. Comparing the distribution of the beta 3 mRNA and the beta 3 isotype shows that the transcription of the beta 3 tubulin gene is cell type specifically repressed during differentiation of individual mesodermal derivatives, from which the dorsal vessel remains transcriptionally active until shortly before hatching. In contrast the beta 3 tubulin protein is detectable in all mesodermal derivatives. The beta 3 tubulin is an excellent marker to study mesoderm differentiation on a regulatory and cellular level using both genetics and molecular biology. In the visceral mesoderm, the expression of the beta 3 tubulin gene is regulated by homeotic gene products, while other transactivators regulate expression in the dorsal vessel and the body wall musculature. In the somatic mesoderm, the beta 3 tubulin allows to visualize myotube formation and insertion into the epidermis. This contact to the epidermal attachment sites (apodemes) induces beta 1 tubulin expression, as can be seen in double staining experiments. We determined a 14bp cis-regulatory enhancer element guiding expression of the beta 1 tubulin gene in these attachment sites. Using the beta 1 and beta 3 tubulin isotypes as markers we started to isolate mutants which are disturbed in muscle formation.

Expression of beta 1 tubulin (beta Tub56D) in Drosophila testis stem cells is regulated by a short upstream sequence while intron elements guide expression in somatic cells.

Stem cell differentiation to mature spermatozoa is a morphogenetic process that is highly dependent on microtubular arrays. In the early, mitotically active stages of spermatogenesis, only the beta 1 tubulin isotype is expressed. Analysis of transgenic flies containing beta 1-lacZ gene fusions revealed that this expression is regulated by sequences located between positions -45 and -191 upstream of the transcription initiation site. Furthermore, beta 1 tubulin is a major component of cyst cells. Expression in these cells is driven by enhancer elements located in the beta 1 tubulin gene intron. These enhancer elements also guide expression in combination with the hsp70 basal promoter. In addition, redundant enhancer elements in the intron drive expression in the testis wall. Our data show that within a single tissue, the male gonad, expression of the beta 1 tubulin gene is under cell-type-specific control mediated by independent cis-acting elements. Therefore in the germ line, control of beta 1 tubulin expression is strictly governed by promoter-proximal elements, while for the somatic parts of the testis, enhancer elements confer less stringent expression control.

An 18-bp element in the 5' untranslated region of the Drosophila beta 2 tubulin mRNA regulates the mRNA level during postmeiotic stages of spermatogenesis.

During Drosophila spermatogenesis transcriptional activity is mainly restricted to premeiotic stages. Translation during sperm morphogenesis, however, proceeds for several days, requiring a high stability for mRNAs translated postmeiotically. We studied expression of the Drosophila beta 2 tubulin gene, which is expressed solely in the male germ line from the primary spermatocyte stage onwards. Cis-acting elements involved in the regulation of mRNA levels were investigated in transgenic fly strains. In adult testes, mRNA amounts from beta 2-lacZ fusion genes in the presence of an 18-bp AT-rich element, termed beta 2DE1, are elevated about threefold. The element is present at about the same position in the 5' untranslated regions of the beta 2 tubulin genes of the distantly related species Drosophila melanogaster and Drosophila hydei. Changing the position of the element on the mRNA reduces the stabilizing effect, while inversion of the beta 2DE1 abolishes its function. The element also acts in a combination with the beta 1 tubulin transcription start site, and the beta 2UE1, which is required to achieve tissue-specific expression. In all experiments done, the comparison of premeiotic with postmeiotic stages strongly implies that this element is involved in regulating mRNA stability. This mRNA stabilizing element acts in a position-independent manner and also on a heterologous mRNA, showing its autonomous functional activity.

Redundant enhancer elements guide beta 1 tubulin gene expression in apodemes during Drosophila embryogenesis.

During Drosophila embryogenesis, the beta 1 tubulin gene (beta Tub56D) is expressed in the CNS and PNS as well as in the apodemes. In this report we determine the regulation of beta 1 tubulin gene expression during formation of the attachment sites of the somatic muscles, the apodemes. The process was analysed in transgenic flies carrying beta 1 enhancer-hsp70 promoter-lacZ fusion constructs. Expression is first detected at late stage 13 and remains until hatching. By deletion analysis of the intron we identified a 14-bp element present in three copies. This element represents a classical enhancer, as it acts on a heterologous promoter. Separate fragments containing the respective elements yield nearly identical expression patterns, and no cooperativity was observed between the three copies. Thus, the expression of the beta 1 tubulin gene in the apodemes is under control of redundant enhancer elements. Double staining for beta 1 tubulin gene expression in apodemes and for beta 3 tubulin gene expression in muscles allowed us to correlate apodeme and muscle formation. Cells of the apodemes that are in contact with their corresponding muscles show expression of the reporter gene as monitored by antibody staining.

During Drosophila embryogenesis the beta 1 tubulin gene is specifically expressed in the nervous system and the apodemes.

We determined the in vivo distribution of the beta 1 tubulin from D. melanogaster using isotype specific antibodies. Maternally expressed beta 1 tubulin is incorporated into mitotic spindles. Later in development a strong expression in the CNS is observed. Furthermore, all chordotonal organs and the apodemes are marked by beta 1 tubulin. Nuclear run-on assays and stage specific in vitro transcription showed a zygotic expression of the beta 1 tubulin gene from the extended germ-band stage onwards. Using the P-element system, we identified several elements; upstream between -2.2 kb and the transcription initiation site, elements for low level expression in the CNS are present. In the intron between +0.44 kb and +2.5 kb enhancer elements are located that drive the expression in the chordotonal organs and the apodemes. Between the start site and +0.44 kb (273 bp) and +2.5 kb and the second exon (315 bp), maternal and CNS enhancers result in full level expression of a lacZ-beta 1 reporter gene. We show, that the beta 1 tubulin gene is very early effector gene starting its expression shortly after the commitment of neuroblast cell fate. This gene offers an excellent model system for the identification of neural and apodeme specific transcription factors.

A purified transcription factor (TIF-IB) binds to essential sequences of the mouse rDNA promoter.

A transcription factor that is specific for mouse rDNA has been partially purified from Ehrlich ascites cells. This factor [designated transcription initiation factor (TIF)-IB] is required for accurate in vitro synthesis of mouse rRNA in addition to RNA polymerase I and another regulatory factor, TIF-IA. TIF-IB activity is present in extracts both from growing and nongrowing cells in comparable amounts. Prebinding competition experiments with wild-type and mutant templates suggest that TIF-IB interacts with the core control element of the rDNA promoter, which is located immediately upstream of the initiation site. The specific binding of TIF-IB to the RNA polymerase I promoter is demonstrated by exonuclease III protection experiments. The 3' border of the sequences protected by TIF-IB is shown to be on the coding strand at position -21 and on the noncoding strand at position -7. The results suggest that direct binding of TIF-IB to sequences in the core promoter element is the mechanism by which this factor imparts promoter selectivity to RNA polymerase I.

Growth-dependent regulation of rRNA synthesis is mediated by a transcription initiation factor (TIF-IA).

Mouse RNA polymerase I requires at least two chromatographically distinct transcription factors (designated TIF-IA and TIF-IB) to initiate transcription accurately and efficiently in vitro. In this paper we describe the partial purification of TIF-IA by a four-step fractionation procedure. The amount or activity of TIF-IA fluctuates in response to the physiological state of the cells. Extracts from quiescent cells are incapable of specific transcription and do not contain detectable levels of TIF-IA. Transcriptionally inactive extracts can be restored by the addition of TIF-IA preparations that have been highly purified from exponentially growing cells. During the fractionating procedure TIF-IA co-purifies with RNA polymerase I, suggesting that it is functionally associated with the transcribing enzyme. We suggest that only those enzyme molecules that are associated with TIF-IA are capable to interact with TIF-IB and to initiate transcription.