Claude Got

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 Laemmlis 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

Summary— 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.

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.

Molecular dissection of a specific nuclear domain: The chromatin region of the ribosomal gene cluster in Xenopus laevis

Molecular dissection of the nuclear domain corresponding to the ribosomal chromatin cluster was investigated. The experimental scheme was based on the ability of restriction enzymes to digest the whole genome without affecting this region (several megabases in length). Such a strategy involved the judicious choice of restriction enzymes, which is possible in Xenopus laevis, where the rDNA sequence is known and the repeated units are organized into one unique cluster. SalI, XhoI, and EcoRV digestion produced frequent cutting of the genome leaving the ribosomal cluster intact. Isolation of the rDNA cluster was confirmed by separation of the digested DNA by pulsed-field electrophoresis. When applied to purified nuclei, this approach allowed the isolation of the ribosomal chromatin cluster under very mild conditions: no cleavages (either enzymatic or mechanical) were detectable. Since the purification scheme depends only on the DNA sequence outside of the rDNA cluster, it permits the obtention of this domain in different functional states. Electron microscopic analysis demonstrated that the domain organization is substantially preserved and maintains its looped organization (the size and the full number of loops were preserved). This purification scheme provides a powerful tool for studying the structure-function relationships within the ribosomal nuclear domain.

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.