The positive transcription factor of the 5S RNA gene proteolyses during direct exchange between 5S DNA sites.

We have examined the association, dissociation, and exchange of the 5S specific transcription factor (TFIIIA) with somatic- and oocyte-type 5S DNA. The factor associates faster with somatic than with oocyte 5S DNA, and the rate of complex formation is accelerated by vector DNA. Once formed, the TFIIIA-5S DNA complex is stable for greater than 4 h in the absence of free 5S DNA, and its dissociation is identical for somatic and for oocyte 5S DNA. In the presence of free 5S DNA, the factor transfers promptly from the complex to the free 5S DNA site. Unexpectedly, the direct exchange of factor between 5S DNA sites leads to proteolysis at the C-terminal arm of TFIIIA.

The C-terminal domain of transcription factor IIIA interacts differently with different 5S RNA genes.

DNase I footprints and affinity measurements showed that the C-terminal arm of Xenopus transcription factor IIIA interacts differently with different Xenopus 5S DNAs, forming three distinct types of transcription factor IIIA-5S DNA complexes: a somatic type, a major-oocyte (and pseudogene) type, and a trace-oocyte type. Site-directed mutagenesis on the major-oocyte 5S gene revealed that somatic-type changes at positions 53, 55, and 56 changed the structure of the transcription factor IIIA-5S DNA complex from major-oocyte to somatic, and a single trace-oocyte change at position 56 caused the change from major-oocyte to trace-oocyte complex. We further show that the somatic-type changes are accompanied by a marked enhancement in the rate of 5S RNA transcription, and we discuss the possible biological relevance of these findings.

Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes.

We describe an in vitro system, based on the Xenopus laevis oocyte supernatant of Glikin et al. (G. Glikin, I. Ruberti, and A. Worcel, Cell 37:33-41, 1984), that packages DNA into minichromosomes with regularly spaced nucleosomes containing histones H3, H4, H2A, and H2B but no histone H1. The same supernatant also assembles the 5S RNA transcription complex; however, under the conditions that favor chromatin assembly, transcription is inhibited and a phased nucleosome forms over the 5S RNA gene. The minichromosomes that are fully loaded with nucleosomes remain refractory to transcriptional activation by 5S RNA transcription factors. Our data suggest that this repression is caused by a nucleosome covering the 5S RNA gene and that histone H1 is not required for regular nucleosome spacing or for gene repression in this system.

Histone H1 represses transcription from minichromosomes assembled in vitro.

We have previously shown that transcription from a Xenopus 5S rRNA gene assembled into chromatin in vitro can be repressed in the absence of histone H1 at high nucleosome densities (one nucleosome per 160 base pairs of DNA) (A. Shimamura, D. Tremethick, and A. Worcel, Mol. Cell. Biol. 8:4257-4269, 1988). We report here that transcriptional repression may also be achieved at lower nucleosome densities (one nucleosome per 215 base pairs of DNA) when histone H1 is present. Removal of histone H1 from the minichromosomes with Biorex under conditions in which no nucleosome disruption was observed led to transcriptional activation. Transcriptional repression could be restored by adding histone H1 back to the H1-depleted minichromosomes. The levels of histone H1 that repressed the H1-depleted minichromosomes failed to repress transcription from free DNA templates present in trans. The assembly of transcription complexes onto the H1-depleted minichromosomes protected the 5S RNA gene from inactivation by histone H1.

Analysis of the chromatin assembled in germinal vesicles of Xenopus oocytes.

We have injected circular DNA, labeled with 32P at a single restriction site, into germinal vesicles of Xenopus laevis oocytes in order to study the nucleosome arrangement on the assembled minichromosomes. Two types of genes were used in these studies, the somatic 5 S RNA gene unit of Xenopus borealis and the histone gene unit of Drosophila melanogaster. We find that injections of labeled DNA alone, at 1 ng DNA per oocyte, results in irregularly spaced nucleosomes and partially supercoiled DNA molecules. However, perfectly spaced nucleosomes are assembled and fully supercoiled DNA is recovered if 5 to 20 nanograms of cold vector DNA is coinjected with the labeled DNA. At the optimum chromatin assembly conditions, the nucleosomes are perfectly spaced with a 180 base-pair periodicity, but they are randomly positioned on the DNA. The assembly of a periodic chromatin structure is accompanied by a dramatic enhancement in the expression of the injected 5 S RNA gene.

Mechanism of chromatin assembly in Xenopus oocytes.

We have analyzed the chromatin assembly reaction catalyzed by the Xenopus oocyte extract (S-150). A 50 S complex is formed upon mixing the 17 S pUC DNA and the S-150. Mature histones are not detected in this complex, which contains relaxed DNA and protein, and generates subnucleosomal 7 S particles upon digestion with micrococcal nuclease. The relaxed nucleoprotein is gradually supercoiled into nucleosomal chromatin in the S-150, via a pathway that requires ATP and is blocked by novobiocin, and this process is accompanied by the appearance of mature histones H3 and H4. Isolated complexes also supercoil in vitro, which implies the complex is a kit that contains histone precursors, as well as topoisomerases and other enzymes required for assembly. We discuss the biological implications of these findings.

Assembly and properties of chromatin containing histone H1.

The Xenopus oocyte supernatant (oocyte S-150) forms chromatin in a reaction that is affected by temperature and by the concentration of ATP and Mg. Under optimal conditions at 27 degrees C, relaxed DNA plasmids are efficiently assembled into supercoiled minichromosomes with the endogenous histones H3, H4, H2A and H2B. This assembly reaction is a gradual process that takes four to six hours for completion. Micrococcal nuclease digestions of the chromatin assembled under these conditions generate an extended series of DNA fragments that are, on average, multiples of 180 base-pairs. We have examined the effect of histone H1 in this system. Exogenous histone H1, when added at a molar ratio of H1 to nucleosome of 1:1 to 5:1, causes an increase in the micrococcal nuclease resistance of the chromatin without causing chromatin aggregation under these experimental conditions. Furthermore, the periodically arranged nucleosomes display longer internucleosome distances, and the average length of the nucleosome repeat is a function of the amount of histone H1 added, when this histone is present at the onset of the assembly process. In contrast, no major change in the length of the nucleosome repeat is observed when histone H1 is added at the end of the chromatin assembly process. Protein analyses of the purified minichromosomes show that histone H1 is incorporated in the chromatin that is assembled in the S-150 supplemented with histone H1. The amount of histone H1 bound to chromatin is a function of the total amount of histone H1 added. We define here the parameters that generate histone H1-containing chromatin with native nucleosome repeats from 160 to 220 base-pairs, and we discuss the implications of these studies.

Chromatin-specific hypersensitive sites are assembled on a Xenopus histone gene injected into Xenopus oocytes.

A cloned histone H4 gene of Xenopus laevis is efficiently transcribed after injection into germinal vesicles of X. laevis oocytes. Deletion analyses indicate that less than 140 base-pairs of 5' flanking sequences and 50 base-pairs of 3' flanking sequences are required for efficient transcription of this gene in Xenopus oocytes. Chromatin footprint analysis by a direct end-label technique reveals discrete DNase I-hypersensitive and micrococcal nuclease-hypersensitive sites at the 5' and 3' boundaries of the gene, which bracket the transcribed region of this minichromosome. The specific chromatin structure assembled around this homologous gene, together with the finding that histone genes of Drosophila melanogaster are not assembled into specific nucleoprotein structures within Xenopus oocytes, strongly suggest that sequence-specific and species-specific factors may be responsible for generating the chromatin-specific hypersensitive sites at the boundaries of active genes.

A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA.

Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.

[Long-term osseous changes in the posterior arch after laminectomy for lumbar stenosis]. / Les remaniements à long-terme de l'arc postérieur restant après laminectomie pour sténose lombaire.

PURPOSE OF THE STUDY: Many studies have indicated favorable results of decompressive surgery for symptomatic lumbar spinal stenosis. However, little is known about the osseous changes that occur at the operative sites. Postacchini in 1992 and Chen in 1994 have studied, only from plain radiographs, osseous changes at the operative sites, and have suggested that bone regrowth possibly affects the neurologic result. The aims of this study were: to assess bone regrowth at the operative site, to compare the bone regrowth rate calculated from plain radiographs and CT-Scan examinations, to determine the effects of bone regrowth on clinical outcome, to investigate the factors promoting the bone regrowth. MATERIAL AND METHOD: 28 patients who underwent decompressive surgery for lumbar spinal stenosis were retrospectively studied with an average follow-up of 8.4 years. In order to evaluate the degree of bone regrowth at the posterior arch, early postoperative radiographs and CT images of the operative sites were compared with those obtained at final follow-up. Bone regrowth at the sites operated upon was evaluated as a percentage of regrowth of the original laminectomy site based upon plain radiographs and CT images. RESULTS: Decompressive lumbar spinal stenosis is responsible for bone regrowth at the operative site in most patients. However, this regrowth was mild, the mean bone regrowth rate evaluated from plain radiographs was 12 per cent in average and the obtained from CT images was 8.2 per cent in average. Changes were found to be predominant at the facet joint level compared to the pedicle level. The evaluation of regrowth obtained from plain films and CT image examinations were compared. Radiographs seem to overestimate bone regrowth. Postoperative spinal instability was statistically significantly associated with new bone development. This variable was the only factor that affected the degree of bone regrowth. No relationship between bone regrowth and clinical outcome was found. DISCUSSION AND CONCLUSION: Natural course of laminectomy defect includes probably new bone formation in most patients. New bone results from gradual regrowth of the laminae and articular processes partially resected at surgery and from coalescence of islets of bone tissue within the tissue filling the laminectomy defect. In the present study bone regrowth rate was moderate but in other ones it was marked. If some factors (like postoperative destabilization) promoting bone regrowth were identified many remain unknown. Factors influencing rapidity of regrowth progression remain also unknown. Patient's intrinsic features such as spinal stenosis characteristics are probably closely related to quantitative and kinetic characteristics of regrowth. Consequences of bone regrowth are also variable: in some cases regrowth may reproduce pathological conditions identical previous ones, in other ones new bone spreads around the dura a mater without any nerves roots compression. Study of bone regrowth requires further research including prospective studies and using a more precise method for the regrowth evaluation.

[Long-term radiologic evolution of coral implanted in cancellous bone of the lower limb. Madreporic coral versus coral hydroxyapatite]. / Evolution radiologique à long terme du corail implanté en os spongieux au membre inférieur. Corail madréporique versus hydroxyapatite de corail.

PURPOSE: The study aimed to compare two successive series of procedure using first pieces of natural madreporic coral, then coralline hydroxyapatite in traumatology. The goal of this work was to evaluate long term radiological features of absorption and influencing factors. MATERIAL: Within six years, 65 pieces were used; only impaction articular fracture on the lateral tibial plateau (31) and calcaneum fractures (23) were included 21 cases completed inclusion criteria. As there were 3 secondary infections, 18 had therefore, an aseptic evolution and were included in this series. METHODS: Absorption was evaluated in five degrees according to the volume of the remaining piece on X-rays. We also took into consideration the position of the pieces and the amount of surfaces in contact with cancellous bone. RESULTS: Among the 18 pieces followed up for more than 19 months (average 40), there were 4 sequestrations (1 tibia, 3 calcaneum), all were natural madreporic coral, the others presented osteointegration. Only natural madreporic coral pieces were absorbed, but never completely and the absorption was slow (3 to 7 years) and never complete. A radiolucent line was present in 7 out of 10 pieces of madreporic coral and none around coralline hydroxyapatite. Three developed favorably. Four were sequestred. Number of surfaces in contact with cancellous bone was correlated with osteointegration. DISCUSSION: The calcium carbonate which makes up the natural madreporic coral pieces is the site of destruction by osteoclasts. The radiolucent line is the specific witness of this biochemical activity. The radiolucent line proceeds sequestrations. No substitute of coralline hydroxyapatite has presented the same sign of peripheral absorption or sequestration. Coralline hydroxyapatite could be the correct coralline material to fill cancellous defect after elevation of an articular depression. In every case, the piece of coral must be fitted in contact with the cancellous bone in the depth of the bone. Conditions of osteoconduction of madreporic coral must be reconsidered. The degree and speed of peripheral absorption must be controlled before weight bearing is allowed.

[Conditions for the consolidation of fractures of the femoral diaphysis after locked intramedullary flexible osteosynthesis]. / Conditions de consolidation des fractures de la diaphyse fémorale après ostéosynthèse centromédullaire flexible verrouillée.

PURPOSE OF THE STUDY: We present here the clinical results achieved with locked intramedullary flexible osteosynthesis (LIFO) system in femoral fractures. The two major advantages of the device are its flexibility an ease of use. MATERIAL AND METHODS: Over a period of 6 years, 42 patients were treated. Two died and 7 were lost for follow-up, leaving 33 cases available for evaluation. Fourteen cases were specifically analysed for callus morphological characteristics: volume, length and width, and compared to 18 cases from a previous series involving 27 intramedullary unmatched appaired nailings. RESULTS: Results in the 33 patients of this series are: no nonunion, 1 osteomyelitis in an open fracture, 4 malunions (2 primary iatrogenic malunions, 2 secondary cases due to a weak fixation). Fractures united at a mean delay of 10 weeks, that is, 4 weeks less than with a nail. Morphological callus study also showed that the LIFO system generates about 25 per cent longer of peripheral new bone formation than a nail. CONCLUSION: A LIFO construct with 3 or even 4 pins yields a shorter healing time than a nail. Furthermore, it generates a callus of 25 per cent longer around the fracture. The four pins construct was found to be as dependable as a locked nail, and minimizes inventory. This technique is indicated in the treatment of all common femoral shaft fractures in adults.