dlarp, a new candidate Hox target in Drosophila whose orthologue in mouse is expressed at sites of epithelium/mesenchymal interactions.

Hox complex genes are key developmental regulators highly conserved throughout evolution. They encode transcription factors that initiate genetic programs of diversified morphogenesis along the anteroposterior embryonic axis. We report the characterization of the novel Drosophila Hox target gene dlarp, isolated from a further screen of a previously described library of genomic DNA fragments associated in vivo with Ultrabithorax proteins. The dlarp spatio-temporal pattern of transcription in wild-type and homeotic mutant embryos is consistent with a positive regulation by Sex combs reduced and Ultrabithorax in the parasegment 2 ectoderm and the abdominal mesoderm, respectively. The teashirt gene product, thought to act in concert with Hox proteins, is also required for the transcriptional control of this target. Search in databases revealed that dlarp has been highly conserved during evolution. The embryonic expression pattern of the mouse orthologue does not support a function downstream of Hox proteins. It is mainly transcribed in neural structures and in developing organs characterized by epithelial-mesenchymal interactions.

nessy, an evolutionary conserved gene controlled by Hox proteins during Drosophila embryogenesis.

From a library of DNA fragments associated with Ultrabithorax protein in vivo, we have isolated nessy, a new Drosophila gene that encodes a putative transmembrane protein conserved in evolution from Caenorhabditis elegans, to human. Zygotic expression occurs transiently in mesectodermal cells at gastrulation, proceeds in mesoderm and endoderm lineages during germ band movements and becomes then restricted to anterior and posterior domains in the visceral mesoderm. The Hox proteins Ultrabithorax, Antennapedia and AbdominalA are likely acting simultaneously to repress nessy in the other parts of the visceral mesoderm.

Isolation of a Drosophila T-box gene closely related to human TBX1.

T-box genes, in all metazoans studied from nematode to man, exist in small gene families. They encode transcription factors with a novel, large, and highly conserved DNA binding domain termed the T-domain. In all cases studied, T-box genes have important developmental roles. Two familial diseases, Holt-Oram syndrome and ulnar-mammary syndrome, were recently shown to be caused by mutations in the human T-box genes TBX5 and TBX3, respectively. T-box genes were first identified in Drosophila and mouse. Two of the three known Drosophila T-box genes show a close sequence homology to mammalian genes. Similarities in the phenotypes of fly and mammalian mutants can be taken as evidence of functional conservation. We report here the isolation of a fourth Drosophila T-box gene, optomotor-blind-related gene-1 (org-1), closely related to mouse and human TBX1. We localized TBX1 to chromosomal band 22q11, confirming a recent report, and discuss TBX1 as a candidate gene for DiGeorge and related syndromes.

Mutations in ccf, a novel Drosophila gene encoding a chromosomal factor, affect progression through mitosis and interact with Pc-G mutations.

We report herein the isolation of ccf, a new gene located in region 82E and essential for Drosophila development. This gene, expressed throughout development, encodes a novel product of 68 kDa which is found in the nucleus during interphase and labels, in a novel pattern, centrosomes and chromosome arms during mitosis. Mutations in ccf give rise to late larvae with small imaginal discs and to adults showing appendages of reduced size, consistent with CCF involvement in cell proliferation. Neuroblast squash analyses show that CCF is required for proper condensation of mitotic chromosomes and, therefore, for progression through mitosis. Furthermore, we observe that adult ccf mutants as well as animals overexpressing CCF during larval stages exhibit homeotic transformations. We also find that mutations in the Pc-G genes Polycomb, polyhomeotic and Enhancer of zeste are enhanced by ccf mutations. Finally, we show that the CCF protein binds to specific sites on polytene chromosomes, many of which are shared with the Posterior sex combs Pc-G protein. Together, these results suggest a role for the CCF protein in the maintenance of chromosome structure during mitosis and interphase.

Distribution of topoisomerase II-mediated cleavage sites and relation to structural and functional landmarks in 830 kb of Drosophila DNA.

The pattern of sites for cleavage mediated by topoisomerase II was determined in 830 kb of cloned DNA from the Drosophila X chromosome, with the objectives of comparing it with mapped structural and functional landmarks and examining if the correlations with such landmarks reported in individual loci can be generalized to a region approximately 100 times longer. The relative frequencies of topoisomerase II cleavage sites in 247 restriction fragments from 67 clones were quantified by hybridization with probes prepared from DNA fragments which abutted all cleavage sites in each clone, selected through the covalently bound topoisomerase II subunit; the specificity and quantitative nature of this method were demonstrated using a plasmid DNA model. The 12 restriction fragments with strong nuclear scaffold attachment (SAR) activity, of which seven possess autonomous replication (ARS) activity, show statistically strong coincidence or contiguity ( P 10 kb; their sensitivity is therefore unlikely to be due to alternating purine-pyrimidine repeats or regions of Z conformation, which are preferred motifs. The hypothesis that they possess intrinsic curvature is consistent with the similarity of their length and spacing to regions of predicted curvature in the 315 kb DNA of Saccharomyces cerevisiae chromosome III and with the reported strong binding preference of topoisomerase II for curved DNA. The topoisomerase II cleavage pattern in this DNA further shows that its relationships to functional properties seen in individual loci, especially to MAR/SAR and ARS activity and to the restricted accessibility of DNA to topoisomerase II in vivo, can be generalized to much longer regions of the genome.

Mapping of genomic DNA loop organization in a 500-kilobase region of the Drosophila X chromosome by the topoisomerase II-mediated DNA loop excision protocol.

The recently developed procedure of chromosomal DNA loop excision by topoisomerase II-mediated DNA cleavage at matrix attachment sites (S. V. Razin, R. Hancock, O. Iarovaia, O. Westergaard, I. Gromova, and G. P. Georgiev, Cold Spring Harbor Symp. Quant. Biol. 58:25-35, 1993; I. I. Gromova, B. Thompsen, and S. V. Razin, Proc. Natl. Acad. Sci. USA 92:102-106, 1995) has been employed for mapping the DNA loop anchorage sites in a 500-kb region of the Drosophila melanogaster X chromosome. Eleven anchorage sites delimiting 10 DNA loops ranging in size from 20 to 90 kb were found within this region. Ten of these 11 anchorage sites colocalize with previously mapped scaffold attachment regions. However, a number of other scaffold attachment regions are found to be located in loop DNA.

SARs, ARSs, and A,T-rich regions evidenced by restriction mapping on an 835-kb DNA fragment from Drosophila.

In Drosophila, sequences anchoring the DNA molecule to the scaffold (SARs) and sequences able to replicate autonomously (ARSs) had been shown to comap on an 835-kb DNA fragment (Brun et al. (1990) Mol. Cell. Biol. 10, 5455-5463). To investigate the question of whether this comapping results from the coincidental recruitment of SARs and ARSs in A,T-rich regions, A,T-rich regions of the 835-kb DNA fragment have been identified by restriction analysis with enzymes recognizing motifs made exclusively of A and T. Within the limits of sensitivity of this approach, the obtained data favor the idea of a noncoincidental recruitment: obviously a SAR and an ARS subpopulation are preferentially localized in the A,T-rich regions, but not every A,T-rich region displays a SAR activity, or an ARS activity, or both, nor are all SARs or ARSs localized in the A,T-rich regions. In addition, the data support the idea that a statistical assessment of base composition using restriction analysis might be developed into a general useful approach to genome organization.

SARs on an 835 kb DNA fragment from the Drosophila genome.

We have investigated the loop organization of a 835 kilobases DNA fragment from the Drosophila genome. This analysis has focused on the periodicity of the distribution of anchoring sequences (SARs) and its relationship to the distribution of A,T-rich regions, transcription units, repeated elements, putative replication origins and topoisomerase II cleavage sites. Altogether, the data support the idea of an active participation of SARs to the structural organization and functioning of this eukaryotic genome.

Relationship between scaffold-attached regions, sequences replicating autonomously in yeast, and a chromosomal replication origin in the Drosophila rDNA.

The potential relationship between anchorage of Drosophila rDNA to a nuclear substructure and replication mechanisms was studied. First, two scaffold-attached regions (SARs) were identified, in the internally transcribed spacer and in the region spanning both the intergenic spacer (IGS) and the externally transcribed spacer (ETS), respectively. These SARs define two possible loops containing the sequence coding for the 18S rRNA and part of that coding for the 28S rRNA, respectively. They also bind yeast scaffolds. Then, the presence of sequences able to promote extrachromosomal replication in yeast (ARSs) was tested. The identified ARSs comap with SARs. The tight relationship between SARs and ARSs was further investigated in the IGS-ETS region which contains a chromosomal replication origin. The topological correlation observed between SARs, ARSs, and a chromosomal replication origin suggests the physical association of the replication origin to the nuclear substructure.

Scaffold-associated regions and repeated or cross-hybridizing sequences on an 800 kilobase DNA stretch of the Drosophila X chromosome.

DNA fragments binding to the scaffold (SARs) have been previously mapped on an 800-kb DNA fragment from the Drosophila X chromosome. Here we have examined whether these 86 DNA fragments bear sequences repeated in the Drosophila genome and/or cross-hybridizing sequences. Twenty-two middle-repeated sequences were localized by hybridizing Southern transfers of representative cloned DNA with total genomic DNA and, reciprocally, by hybridizing Southern transfers of total genomic DNA with this cloned DNA. Seventy-nine out of the 86 SAR-containing fragments appeared to be distinct from these 22 repeated motifs. Therefore, SARs, in their vast majority, are not members of middle-repeated sequence families. However, the 22 middle-repeated sequences were shown to be significantly located in the near vicinity of SARs. Hybridizations were also performed between SAR-containing DNA fragments, either at a high or at a low stringency. At a high stringency, the 86 SAR-containing DNA fragments did not cross-hybridize. However, at a low stringency, a complex hybridization network was evidenced. These nucleotide sequence similitudes support the idea that the various SARs may play common roles.

Studies of an 800-kilobase DNA stretch of the Drosophila X chromosome: comapping of a subclass of scaffold-attached regions with sequences able to replicate autonomously in Saccharomyces cerevisiae.

We have previously mapped scaffold-attached regions (SARs) on an 800-kilobase DNA walk from the Drosophila X chromosome. We have also previously shown that the strength of binding, i.e., the ability of SARs to bind to all nuclear scaffolds or only to a fraction of them varied from one SAR to another one. In the present study, 71 of the 85 subfragments that bind scaffolds and 38 fragments that do not bind scaffolds were tested for their ability to promote autonomous replicating sequence (ARS) activity in Saccharomyces cerevisiae. Sixteen SAR-containing fragments from the chromosome walk were also examined for association to yeast nuclear scaffolds in vitro. All identified ARSs (a total of 27) were present on SAR-containing fragments, except two, which were adjacent to SARs. There is thus a correlation between ARS and SAR activities, and this correlation defines a SAR subclass. Moreover, the presence of an ARS on a DNA fragment appeared to be highly correlated with the strength of binding. The binding activity was highly conserved from Drosophila melanogaster to yeast. These data suggest that Drosophila DNA sequences responsible for binding to components of the nuclear scaffold from either D. melanogaster or yeast may be involved in the process of heterologous extrachromosomal replication in yeasts.

Supragenic loop organization: mapping in Drosophila embryos, of scaffold-associated regions on a 800 kilobase DNA continuum cloned from the 14B-15B first chromosome region.

The supragenic loop organization of the Drosophila genome was investigated on a 800 kilobase (kb) DNA continuum from the 14B-15B first chromosome region. Nuclear scaffolds from 0-18 hr embryos were prepared with Laemmli's low-salt, detergent procedure and digested with restriction enzymes. Scaffold-associated regions (SARs) were mapped by probing Southern transfers of total, scaffold-associated and free DNA with a set of 70 recombinant phages overlapping the investigated genomic region. In all, 85 restriction fragments showed association to scaffolds. 12 of them were present in the majority of scaffolds. They bore strong SARs organizing the DNA molecule as consecutive loops with sizes ranging from 15 to 115 kb. 44 were present in only a fraction of scaffolds. They contained weak SARs subdividing the basic loops into smaller ones. 29 additional restriction fragments were present in a very small fraction of scaffolds. The position of SARs with respect to transcribed regions was investigated. Strong SARs appeared to be located on untranscribed DNA and to frame transcription units. In contrast, at least some weak SARs were shown to comap with transcribed regions or to reside within characterized transcription units. Statistical analyses established that strong and weak SARs were periodically positioned on the DNA continuum and that there was a potential contact point between scaffolds and the DNA continuum every 11 kb, or multiples thereof. Implications for SAR role(s) are discussed.

Developmental expression pattern of a 800 kb DNA continuum cloned from the Drosophila X chromosome 14B-15B region.

The expression throughout development of a long DNA segment from the Drosophila genome was investigated. A near continuum of 800 kb DNA was cloned by extending a 90 kb previous chromosome walk, spanning the rudimentary locus (r), and by initiating 2 chromosome walks from an isolated DNA clone located next to the r locus. Hexanucleotide-primed single strand cDNAs were synthesized from poly(A)+ RNAs prepared from staged embryos, larvae, pupae, males, females, heads from adults and thoraces plus abdomens from adults. The representativity of the cDNAs with regard to the whole length and the molar abundance of a selected gene mRNA was checked. These cDNAs were then used to probe Southern transfers of the cloned DNA after digestion with combinations of restriction enzymes. The limits of the expressed parts of the cloned DNA and their developmental profiles were then deduced. Data show that, in contrast to what was known on the basis of genetic analysis, this region of the Drosophila genome is densely transcribed. Numerous parts of the cloned DNA are found to be stage-, or sex- or territory-specifically expressed. Data are consistent with a view of the organization of this representative part of the Drosophila genome according to which the expressed regions tend to be clustered, whereas no tendency for transcription units displaying comparable specific spatial, temporal or sex expression patterns to be arranged in groups is noted.

Structural organization and expression of amplified chromosomal sequences, which include the rudimentary gene, in cultured Drosophila cells resistant to N-(phosphonacetyl)-L-aspartate.

We have used 160 kilobases of cloned Drosophila genomic DNA from the rudimentary (r) region to examine the organization of amplified DNA in Drosophila cells resistant to 10 mM N-(phosphonacetyl)-L-aspartate (PALAr cells) obtained by stepwise selection. Evidence for the direct tandem linkage of the amplified sequences is presented. The pattern and intensity of amplified bands as well as the presence of novel junctions in the DNA sequence of PALAr cells indicate that there are two types of units of 150 and 120 kilobases long. The sequence of the smaller unit is entirely included within the larger one. The longer of the two units is present twice while the shorter one is amplified eightfold as compared to the level of the relevant DNA sequences in the wild-type. These data are consistent with a model in which successive crossing-over events over several cell cycles lead to amplification of the selected r gene and flanking sequences. However, these data can also be accounted for by a totally different mechanism in which multiple copies of DNA are generated by rolling circle replication. Transcription units other than the r gene are present within the 150 kilobase region of amplified DNA. These are found to be overexpressed in PALAr cells, though some transcripts are underrepresented relative to the copy number of the corresponding coding sequences.

An immunological approach to quantitate RNA polymerases in plant cell extracts.

A polyclonal antiserum was raised against highly purified RNA polymerase II from soybean embryos. Pure RNA polymerase II was fractionated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose sheets, incubated with the immune antiserum and then with iodinated protein A. Autoradiograms showed that the immune antiserum recognized all subunits of RNA polymerase II. Subunits 42, 27 and 16 kdalton were particularly reactive. Application of this transfer technique to protein extracts from soybean embryos or from cultured soybean cells allowed the identification of subunits of RNA polymerase II in the extracts. Analysis of the staining of the bands on the autoradiograms for increasing amounts of pure RNA polymerase II demonstrated that the transfer was quantitative, so that standard curves could be drawn to estimate the unknown amounts of enzyme in the extracts.

Immunological estimation of changes in the absolute amounts of nuclear RNA polymerases in strictly auxin-requiring, cultured soybean cells upon addition of 2,4-dichlorophenoxyacetic acid.

Soybean cells were subcultured in the presence of 4 µM 2,4-dichlorophenoxyacetic acid (2,4-D;=stock cells), or transferred and subcultured in a culture medium depleted of 2,4-D (=control cells), then reincubated with 4 µM 2,4-D (=treated cells). Control cells responded to 2,4-D by resuming cell division and DNA, RNA and protein synthesis at levels which were comparable to those of stock cells. This study system was used to investigate auxin effects on RNA polymerases. Both activities of RNA polymerases and their absolute amounts in extracts from stock, control and treated cells were determined, using the in-vitro assy of transcription of denatured calf thymus DNA and the specific quantitative immunological method reported in Miassod and Got (1984, Planta 162, 427-434), respectively. The data showed that the levels of enzymes did not remain constant in the stock cells during the subculture cycle, that auxin treatment enhanced, by a factor of two to four, the amounts of RNA polymerase I and II, expressed as percents of total proteins of soybean cells, and that this increase was not necessarily reflected by a parallel increase of the activity measured in the in-vitro test.

Selective transcription of a cloned cauliflower mosaic virus DNA fragment in vitro by soybean RNA polymerase II in the presence of dinucleotide primers.

Transcription of a cloned cauliflower mosaic virus (CaMV) DNA fragment (plasmid pCa 8) was studied at a low enzyme: DNA ratio. Preincubation with purine nucleoside triphosphates leads to essentially random transcription, while in the presence of a dinucleoside monophosphate and a purine nucleoside triphosphate in the preincubation medium certain combinations prime preferential transcription of the eucaryotic moiety of the chimeric plasmid. Characterisation of transcription primed by the most efficient combination, ApG + ATP, shows that a low enzyme: DNA ratio is absolutely essential for selective initiation. Interestingly the presence of the eucaryotic insertion is essential for the transcription of vector sequences. Analysis of RNA primed by ApG + ATP and of short chains synthesised in the presence of the GTP analogue 3'-OMeGTP shows a high degree of selectivity of transcription initiation sites. Hybridisation of primed RNA to restriction fragments of pCa8 shows that initiation occurs within a limited region of the inserted CaMV fragment.

Studies on the methylation of cytoplasmic ribosomal RNA from cultured higher plant cells.

The methylation of cytoplasmic ribosomal RNA of cultured sycamore cells (Acer pseudoplatanus L.) was investigated. Labelled 17-S and 26-S rRNA were prepared from cells that had been incubated with either [32P]phosphate, [Me-3H]methionine or [Me-14C]methionine. Ion-exchange resin chromatography of 0.3 M KOH or 1 M HCl hydrolysates and two-dimensional chromatographic analyses of phosphodiesterase plus phosphatase digests of 17-S and 26-S rRNA were performed. 17-S and 26-S rRNA contain 49 and 91 methyl groups per molecule, respectively. These values were verified in sevemral ways. The high degree of methylation of sycamore rRNA, particularly for the 26-S rRNA, contrasts with the situation in all other investigated organisms. Several methylated bases were identified. 7-Methylguanine and 5-methylcytosine both occur in 17-S and 26-S rRNA. N6-Methyladenine and N6,N6-dimethyladenine are restricted to the 17-S rRNA while 3-methyluracil and 1-methyladenine occur in the 26-S rRNA. One hypermodified uridine was also tentatively identified in the small rRNA. In 17-S rRNA, there is one copy of 7-methylguanine, N6-methyladenine and hypermodified uridine and two copies of N6,N6-dimethyladenine. 3-Methyluracil, 1-methyladenine and 5-methylcytosine occur twice, twice and three times, respectively, in 26-S rRNA. 7-Methylguanine and 5-methylcytosine are only in submolar amounts in the 26-S and 17-S rRNA, respectively. There are 40 +/- 2 and 83 +/- 3 2'-O-methylriboses per 17-S and 26-S rRNA molecule, respectively. In addition to the four 2'-O-methylnucleosides, one 2'-O-methylpseudouridine is present in the 17-S rRNA. Several lines of evidence argues for a non-random distribution of the methylriboses. In particular, one and seven Nm-Nm-Np structures occur in the 17-S and 26-S rRNA, respectively. The data are discussed comparatively with the methylation pattern of Escherichia coli, yeast and HeLa cell rRNA.

Partial base-methylation and other structural differences in the 17 S ribosomal RNA of sycamore cells during growth in cell culture.

Sycamore (Acer pseudoplatanus L.) cytoplasmic rRNA was investigated in rapidly dividing cells, cells starting mitosis after the lag phase of growth (4 days) induced by deconditioning of the culture medium and also in growth-arrested cells from 10 day-old cultures deprived of exogenous auxin (i.e. exponential, early exponential and 2,4-dichlorophenoxyacetic acid (2,4-D)-deprived cultures). rRNA was extracted and purified from mixed 14C-labelled exponential cultures and 3H-labelled early exponential cultures. A 14C-labelled exponential culture and a 3H-labelled 2,4-D-deprived culture were analyzed in the same way. The 17 S rRNA molecules from both early exponential and 2,4-D-deprived cultures displayed a lower electrophoretic mobility on polyacrylamide gels than those from exponential cultures. Alkaline and acid hydrolysates of purified 17 S rRNA labelled on the phosphate groups or the methyl groups were analyzed on ion-exchange resins. There was no change in the extent of ribose methylation of the molecule from the three different cultures. However, the base methylation of the 17 S rRNA was decreased in early exponential cultures and in 2,4-D-deprived cultures. Part of the molecules synthesized in early exponential cultures specifically lacked 7-methylguanine, N6-methyladenine and N6,N6-dimethyladenine. The possible significance of these changes in the 17 S rRNA were discussed.