David Schlessinger

Fragile X genotype characterized by an unstable region of DNA

DNA sequences have been located at the fragile X site by in situ hybridization and by the mapping of breakpoints in two somatic cell hybrids that were constructed to break at the fragile site. These hybrids were found to have breakpoints in a common 5-kilobase Eco RI restriction fragment. When this fragment was used as a probe on the chromosomal DNA of normal and fragile X genotype individuals, alterations in the mobility of the sequences detected by the probe were found only in fragile X genotype DNA. These sequences were of an increased size in all fragile X individuals and varied within families, indicating that the region was unstable. This probe provides a means with which to analyze fragile X pedigrees and is a diagnostic reagent for the fragile X genotype.

X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein.

Ectodermal dysplasias comprise over 150 syndromes of unknown pathogenesis. X–linked anhidrotic ectodermal dysplasia (EDA) is characterized by abnormal hair, teeth and sweat glands. We now describe the positional cloning of the gene mutated in EDA. Two exons, separated by a 200–kilobase intron, encode a predicted 135–residue transmembrane protein. The gene is disrupted in six patients with X;autosome translocations or submicroscopic deletions; nine patients had point mutations. The gene is expressed in keratinocytes, hair follicles, and sweat glands, and in other adult and fetal tissues. The predicted EDA protein may belong to a novel class with a role in epithelial–mesenchymal signalling.

CONSERVATION OF RIBOSOMES DURING BACTERIAL GROWTH.

Two bands are observed when ribosomes from extracts of Escherichia coli are examined by density-gradient centrifugation in cesium chloride solution containing sufficient magnesium ions. The denser is called A and the less dense B. The B band diminishes and A augments as the concentration of magnesium ions is reduced below 0·04 M until at 0·002 M only A remains; below this concentration, particles in the A band are unstable and appear to release free RNA. Isolated 30 s and 50 s ribosomes form bands in the A region, but particles re-isolated from the A band sediment at 23 s and 42 s. The particles in the A band seem to be deficient in protein relative to normal ribosomes. The B band probably contains 70 s ribosomes, although this has not been definitely shown. An experiment was performed to learn whether parts of the 30 s and 50 s ribosomes undergo exchange or renewal during bacterial growth. A culture of E. coli uniformly labeled with heavy isotopes and phosphorus-32 was transferred to light non-radioactive medium in which growth was allowed to continue. At one and three generations after transfer, 30 s and 50 s ribosomes were isolated from samples of the culture and examined by density-gradient centrifugation. The dense 23 s and 42 s particles did not undergo any significant decrease in density as the bacteria multiplied in light medium, nor was phosphorus-32 seen to accumulate in light ribosomes much beyond a small amount found at the first generation. We conclude that the 23 s and 42 s nucleoprotein subunits of 30 s and 50 s ribosomes are conserved during bacterial growth.

Polyribosome metabolism in Escherichia coli: I. Extraction of polyribosomes and ribosomal subunits from fragile, growing Escherichia coli☆

Abstract 1. (1) Two techniques are used that yield cultures of Escherichia coli growing actively in a fragile form: growth of a sucrose-dependent mutant in low concentration of sucrose and growth of wild-type strains in 0.5 m -Na2SO4. 85 to 100% of the cellular RNA is released from such cells chilled with frozen 20% sucrose and lysed in 0.5% sodium deoxycholate, 6 m m -MgSO4, 40 m m -NaCl, 0.3 m m -chloramphenicol, 10 m m -Tris-HCl (pH 7.5). 2. (2) In sucrose gradient centrifugation of the lysates, few if any ribosomes sediment as 70 s particles. About half the total cellular ribosomes appear as 30 s and 50 s subunits. The rest (60 to 65% in the sucrose-dependent mutant; 48 to 53% in other strains) sediment in polyribosomes, moving from 100 s up to ~ 850 s (1.8 times as fast as T7 bacteriophage). Across the polyribosomal profile about the same amount of ribosomes appears in each gradient fraction. This indicates that the number of polyribosomes containing n ribosomes is roughly proportional to 1 n . DNase does not affect the sedimentation profiles, and when cellular DNA has been prelabeled, none appears in the polyribosomes. Thus, the polyribosomes observed are not DNA-dependent aggregates. 3. (3) All the polyribosomes dissociate in 5 × 10−5 m -Mg2+ to subunits and rapidly labeled messenger RNA. RNase treatment converts all the polyribosomes to material that sediments at 70 s, bearing pulse-labeled nascent protein; no polyribosomes appear as 30 s and 60 s subunits. 4. (4) The 30 s and 50 s subunits observed in gradients do not form 70 s “monomers” even in 0.01 m -Mg2+. They bear no nascent protein in extracts of pulselabeled cells. If cells are chilled quickly, but in absence of chloramphenicol, more subunits appear. At the same time, pulse-labeled nascent protein is released to the supernatant phase. Completion of a protein chain may therefore entail the release of a 30 s and 50 s subunit from a messenger RNA. 5. (5) Since slow chilling of cells in absence of chloramphenicol or mild shearing of lysates leads to the appearance of 70 s ribosomes and apparently degraded smaller polyribosomes, earlier patterns which showed many 70 s particles in lysates prepared without similar precautions probably reflected considerable degradation of polyribosomes. 6. (6) Extraction of cells in various media or after pre-treatment with chloramphenicol for five minutes gives nearly identical gradient patterns. The same profile is obtained even in the absence of chloramphenicol if cells are lysed quickly enough—for example, when cultures are harvested by filtration and lysed within two seconds. The characteristic pattern of transiently inactive subunits and heterodisperse polyribosomes may be close to the actual distribution of ribosomes in the living cell.

Polyribosome metabolism in Escherichia coli: II. Formation and lifetime of messenger RNA molecules, ribosomal subunit couples and polyribosomes

During a continuous label with [3H]uracil of fragile Escherichia coli cells in exponential growth, all or very nearly all the hybridizable mRNA of the cells can be observed in polyribosomes during zonal sedimentation in sucrose gradients. In contrast, all the newly formed rRNA appears to be free of polyribosomes until it is part of complete ribosomal subunits. The separation of the two major classes of newly formed RNA permits the direct observation of the growth with time of mRNA molecules, of ribosomal subunits and of polyribosomes. A complete chain of ribosomal RNA forms in one to two minutes; completion of either a 30 s or 50 s ribosome requires a minimum of about five minutes more. The specific activities of free ribosomal subunits and those that are present in polyribosomes increase at identical rates, consistent with very rapid exchange of the two classes. Before any new subunits are finished, the 3H-labeled RNA in polyribosomes is the cellular mRNA, which can therefore be extracted and observed separated from ribosomal RNA. Assuming that their sedimentation rates in sucrose gradients are related to molecular weights as are those of ribosomal rRNA and that no degradation occurs dining extraction, most of the molecules of mRNA are large enough to code for only one or two protein chains of 30,000 molecular weight. The kinetics of entry of all the new mRNA into polyribosomes is in agreement with the view that a polyribosome forms as the corresponding messenger RNA is synthesized, over a period of at least a minute. 3% of the total cellular RNA in these cultures (doubling time 120 minutes) is mRNA, with an average chemical lifetime of 11 to 12·5 minutes.

Classification of Polyene Antibiotics According to Chemical Structure and Biological Effects

Fourteen polyene antibiotics and six of their semisynthetic derivatives were compared for their effects on potassium (K+) leakage and lethality or hemolysis of either Saccharomyces cerevisiae or mouse erythrocytes. These polyene antibiotics fell into two groups. Group I antibiotics caused K+ leakage and cell death or hemolysis at the same concentrations of added polyene. In this group fungistatic and fungicidal levels were indistinguishable. Group I drugs included one triene (trienin); tetraenes (pimaricin and etruscomycin); pentaenes (filipin and chainin); one hexaene (dermostatin); and one polyene antibiotic with unknown chemical structure (lymphosarcin). Group II antibiotics caused considerable K+ leakage at low concentrations and cell death or hemolysis at high concentrations. The fungistatic levels were clearly separable from fungicidal. This group included the heptaenes (amphotericin B, candicidin, aureofungin A and B, hamycin A and B), and five of their semisynthetic derivatives (amphotericin B methyl ester, N-acetyl-amphotericin B, hamycin A and B methyl esters, and N-acetyl-candicidin). Nystatin, classified as a tetraene, and its derivative, N-acetyl nystatin, also were in this group.

Sequence of human glucose-6-phosphate dehydrogenase cloned in plasmids and a yeast artificial chromosome

The sequence of 20,114 bp of DNA including the human glucose-6-phosphate dehydrogenase (G6PD) gene was determined. The region included a prominent CpG island, starting about 680 nucleotides upstream of the transcription start site, extending about 1050 nucleotides downstream of the start site, and ending just at the start of the first intron. The transcribed region from the start site to the poly(A) addition site covers 15,860 bp. The sequence of the 13 exons agreed with published cDNA sequence and for the 11 exons tested, with the corresponding sequence in a yeast artificial chromosome (YAC). The latter confirms YAC cloning fidelity at the DNA sequence level. Sixteen Alu sequences constitute 24% of the total sequence tract. Four were outside the borders of the mRNA transcript of the gene; all the others were found in a large (9858 bp) intron between exons 2 and 3. Two Alu clusters each contain Alus lying between the monomers of another.

Mapping human chromosomes by walking with sequence-tagged sites from end fragments of yeast artificial chromosome inserts.

Sequence-tagged sites (STSs) derived from end fragments of chromosome-specific yeast artificial chromosomes (YACs) can facilitate the assembly of an overlapping YAC/STS map. Contigs form rapidly by iteratively screening YAC collections with end-fragment STSs from YACs that have not yet been detected by any previous STS. The map is rendered rapidly useful during its assembly by incorporating supplementary STSs from genes and genetic linkage probes with known locations. Methods for the systematic development and testing of the end-fragments STSs are given here, and a group of 100 STSs is presented for the X chromosome. The mapping strategy is shown to be successful in simulations with portions of the X chromosome already largely mapped into overlapping YACs by other means.

Yeast artificial chromosomes: tools for mapping and analysis of complex genomes

Libraries of yeast artificial chromosomes (YACs) are representative of complex genomes, and typical YACs contain single fragments of DNA that are up to a megabase or more in size, are stable during growth, and are faithful to genomic DNA. Such YACs may permit (1) the use of complete gene units for a number of research and medical purposes; and (2) the assembly of chromosome-sized contigs in a single map that unifies genetic and physical data.

Involvement of oxidative damage in erythrocyte lysis induced by amphotericin B.

Lysis of human erythrocytes induced by amphotericin B was retarded when the oxygen tension of the incubation mixture was reduced or when the antioxidant catalase was added; lysis was accelerated when cells were preincubated with the prooxidant ascorbate. In the atmosphere of reduced oxygen tension, the erythrocytes containing carboxyhemoglobin lysed at a slower rate than did the cells containing oxyhemoglobin. Consistent with a role for oxidative damage in lysis, the mixture of erythrocytes and amphotericin B showed an increase in malonyldialdehyde, the product of peroxidation, which paralleled the progression of hemolysis. In contrast, the permeabilizing effect of amphotericin B, measured as a decrease in intracellular K+, was not affected by changes in oxygen tension, catalase, or ascorbate treatment. These results imply that oxidant damage is involved in the lytic, but not in the permeabilizing, action of amphotericin B.