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Biomedical subjects

M G Sargent

Publications and source records attributed to M G Sargent.

At least 19 recordsLinked to original sources
PubMed1995

Induction of the prospective neural crest of Xenopus.

The earliest sign of the prospective neural crest of Xenopus is the expression of the ectodermal component of Xsna (the Xenopus homologue of snail) in a low arc on the dorsal aspect of stage 11 embryos, which subsequently assumes the horseshoe shape characteristic of the neural folds as the convergence-extension movements shape the neural plate. A related zinc-finger gene called Slug (Xslu) is expressed specifically in this tissue (i.e. the prospective crest) when the convergence extension movements are completed. Subsequently, Xslu is found in pre- and post-migratory cranial and trunk neural crest and also in lateral plate mesoderm after stage 17. Both Xslu and Xsna are induced by mesoderm from the dorsal or lateral marginal zone but not from the ventral marginal zone. From stage 10.5, explants of the prospective neural crest, which is underlain with tissue, are able to express Xslu. However expression of Xsna is not apparently specified until stage 12 and further contact with the inducer is required to raise the level of expression to that seen later in development. Xslu is specified at a later time. Embryos injected with noggin mRNA at the 1-cell stage or with plasmids driving noggin expression after the start of zygotic transcription express Xslu in a ring surrounding the embryo on the ventroposterior side. We suggest this indicates (a) that noggin interacts with another signal that is present throughout the ventral side of the embryo and (b) that Xslu is unable to express in the neural plate either because of the absence of a co-inducer or by a positive prohibition of expression. The ventral co-inducer, in the presence of overexpressed noggin, seems to generate an anterior/posterior pattern in the ventral part of the embryo comparable to that seen in neural crest of normal embryos. We suggest that the prospective neural crest is induced in normal embryos in the ectoderm that overlies the junction of the domains that express noggin and Xwnt-8. In support of this, we show animal cap explants from blastulae and gastrulae, treated with bFGF and noggin express Xslu but not NCAM although the mesoderm marker Xbra is also expressed. Explants treated with noggin alone express NCAM only. An indication that induction of the neural plate border is regulated independently of the neural plate is obtained from experiments using ultraviolet irradiation in the precleavage period. At certain doses, the cranial crest domains are not separated into lateral masses and there is a reduction in the size of the neural plate.

Amino Acid Sequence↗
PubMed1994

Control of cell behavior during vertebrate development by Slug, a zinc finger gene.

Slug, a vertebrate gene encoding a zinc finger protein of the Snail family, is expressed in the neural crest and in mesodermal cells emigrating from the primitive streak. Early chick embryos were incubated with antisense oligonucleotides to chick Slug. These oligonucleotides specifically inhibit the normal change in cell behavior that occurs at the two sites in the emerging body plan in which the gene is expressed. This change, which is the transition from epithelial to mesenchymal character, occurs at the formation of mesoderm during gastrulation and on emigration of the neutral crest from the neural tube.

Amino Acid Sequence↗
PubMed1992

Cloning and developmental expression of Sna, a murine homologue of the Drosophila snail gene.

The genetic analysis of dorsoventral patterning in Drosophila has identified a zinc-finger gene, snail, that is required for mesoderm formation. The cloning and nuclease protection analysis of a Xenopus homologue of this gene has suggested a possible role in the mesoderm of vertebrates. Here, we describe the cloning of a murine homologue of snail, Sna, and in situ hybridisation studies of its developmental expression. Sequence analysis reveals substantial conservation of the second to fifth zinc fingers, but not of the first zinc finger in the Sna gene. Expression occurs in the ectoplacental cone, parietal endoderm, embryonic and extraembryonic mesoderm, in neural crest and in condensing precartilage. Based on the timing and spatial restriction of expression in embryonic mesoderm, we suggest that Sna might be required for the early development of this tissue, as is the case for its Drosophila counterpart. In addition, we propose that Sna might have an analogous role in the development of neural crest. The expression in condensing precartilage indicates that this gene also has a later function in chondrogenesis.

Amino Acid Sequence↗
PubMed1990

Identification in Xenopus of a structural homologue of the Drosophila gene snail.

We have cloned a Xenopus cDNA that is related to snail, a gene that is required for mesoderm formation in Drosophila. The cDNA encodes a protein that contains five zinc-fingers that closely resemble those of snail. In the non-canonical parts of the DNA-binding loop, there is almost 90% homology between snail and xsna. The corresponding mRNA (xsna) is expressed strongly at the start of zygotic transcription simultaneously with the transcription factor EF1 alpha. In early gastrulae, xsna is equally distributed between the dorsal and ventral halves of the equatorial zone. The possibility that the capacity to synthesise xsna is more localised before the start of zygotic transcription has been investigated by culturing fragments of stage 8 embryos until xsna is synthesised. The capacity to synthesise xsna at stage 8 is located principally in the dorsal half of the equatorial zone. A small amount of maternal xsna is localised in the vegetal hemisphere before zygotic transcription starts. xsna is not present in isolated animal caps but can be induced by the mesoderm-inducing factors XTC-MIF and bFGF. Synthesis of xsna does not occur autonomously in dispersed cells but is restored when cells reaggregate in the presence of calcium and magnesium.

Animals↗
PubMed1987

Fiftyfold amplification of the Lowry protein assay.

The blue product of the Lowry et al. (1951, J. Biol. Chem. 193, 265-275) reaction interacts with malachite green (MG), inducing a change in the visible light spectrum. At A690 nm the absorbance of malachite green solutions increases 10-fold in the presence of Lowry blue (LB). Under the optimum conditions, 0.01 A700 nm unit of Lowry blue produces a change in A690 nm unit of malachite green of 0.5 and the delta A690 nm is a linear function of Lowry blue concentration. Conditions under which this 50-fold amplification can be exploited to detect less than 100 ng of protein (or 4 micrograms X ml-1) are described. A number of chemicals including sodium dodecyl sulfate can interfere with the assay but a strategy has been devised to overcome these problems. Amplification of the Lowry assay appears to involve a cooperative interaction between malachite green and the Lowry blue product such that about 23 molecules of malachite green undergo a spectral shift per molecule of a model reactant such as tyrosine. Malachite green can be used to amplify the molybdenum blue signal obtained in other assays. Less than 10 pmol of tyrosine can be detected using this procedure. Lowry blue also interacts with auramine O, giving a large increase in A500 nm and a 40-fold amplification of the LB signal. As with malachite green, there is a cooperative interaction between auramine O and LB. About 72 molecules of auramine O undergo a spectral shift per molecule of tyrosine. The product of this reaction is also fluorescent and could be exploited in a protein assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzophenoneidum↗
PubMed1986

Identification of a specific membrane-particle-associated DNA sequence in Bacillus subtilis.

After the Bacillus subtilis nucleoid was dissected with restriction endonucleases, a specific DNA sequence from the purA region was isolated in a particulate form that probably originated from the cell membrane. Precise definition of the binding region within this sequence was achieved by a novel procedure based on a previously reported observation that additional copies of the binding region, introduced into the chromosome using an integrative plasmid, were also predominantly particle bound. Subsections of the original plasmid insertion were cloned into the integrative plasmid and introduced into B. subtilis, in which they became tandemly reiterated under appropriate selective conditions. HaeIII sites in the vector, flanking each insertion, were used to excise the latter for subsequent tests of particle association. Examination of 10 strains containing subsections of the original 5.2-kilobase-pair region showed that the binding region was confined to 283 base pairs. This was confirmed by dissection in vitro of a larger, isolated, particle-bound sequence. The nucleotide sequence of a 1,300-base-pair region that contained this site was determined. The entire region had a notably high A + T content and was deficient in open reading frames for transcription.

Bacillus subtilis↗
PubMed1985

Potentiation of a nucleolytic activity in Bacillus subtilis.

In several strains of Bacillus subtilis extensive breakdown of chromosomal DNA may be potentiated by osmotic lysis of protoplasts. At its most severe, in strains originating from Farmer & Rothman's thymine auxotroph, the rate of DNA breakdown was greater than 50% per hour at 40 degrees C. The rate of DNA breakdown in most other strains tested was approximately 5% per hour except for SP beta- strains, in which the rate of DNA breakdown was only 0.3%. DNA degradation was attributed to relaxation of control of a nuclease specified by the prophage of SP beta or a related phage. The most potent nuclease in lysates was an ATP-activated protein of Mr 280 000. Derivatives of Farmer and Rothman's strain containing integrated plasmids had the highest rate of DNA degradation. Although the chromosome was completely destroyed, covalently closed circular plasmids were generated from the integrated sequence. These showed massive deletions of the B. subtilis part of the integrated plasmid but the vector sequence remained intact. The nucleolytic activity therefore appears to recognize specific sequences in B. subtilis DNA. We suggest that activation of SP beta genes during development of competence may be a cause of deletion of cloned genes in the early stages of establishment of cloned sequences.

Adenosine Diphosphate↗
PubMed1985

Amplification of a major membrane-bound DNA sequence of Bacillus subtilis.

A membrane-bound DNA sequence from Bacillus subtilis was subcloned into a plasmid which can replicate in Escherichia coli but not in B. subtilis. This plasmid hybridized with an 11-kilobase HindIII fragment which is the major particle-bound fragment in lysates treated with HindIII. The plasmid integrated into the B. subtilis chromosome at the region of homology, conferring chloramphenicol resistance on the recipient. The inserted resistance was mapped close to purA by using the generalized transducing phage AR9. In one chloramphenicol-resistant strain, the pMS31 region was repeated at least 20 times. A large proportion of the copies of the cloned region were present in the particle fraction, indicating that the capacity to bind this region of the chromosome was substantially in excess of the normal dose of the region. The structure of the particle-bound region was sensitive to ionic detergents and high salt concentrations but was not greatly affected by RNase or ethidium bromide. The basis of a specific DNA-membrane interaction can now be studied by using the amplified region, without the complications of sequences required for autonomous plasmid replication.

Bacillus subtilis↗
PubMed1985

Membrane particles from Escherichia coli and Bacillus subtilis, containing penicillin-binding proteins and enriched for chromosomal-origin DNA.

Rapid-sedimenting DNA-membrane complexes were obtained from both Bacillus subtilis and Escherichia coli by a method involving gentle lysis followed by restriction enzyme digestion and sucrose gradient fractionation. These complexes were substantially enriched in chromosomal origin DNA, and in B. subtilis, the complexes were enriched in penicillin-binding proteins relative to that of the total membrane. Such complexes may represent procaryotic membrane domains which are topographically and functionally distinct.

Bacillus subtilis↗
PubMed1985

How do bacterial nuclei divide?

The mechanism of nuclear division remains unknown. Interactions with the cell surface may play a crucial role both in nuclear organization and division.

Bacteria↗
PubMed1984

Characterization of the replication terminus of the Bacillus subtilis chromosome.

DNA in the terminal region of the chromosome of Bacillus subtilis was labelled by a procedure in which cells in sporulation inducing conditions were pulse-labelled with [3H]thymidine and then treated with p-hydroxyphenylazouracil, an inhibitor of DNA synthesis. The labelled DNA in isolated spores yielded a small number of restriction fragments. About 14 EcoRI fragments with a total length of 80 kb were labelled in a 2.5 min pulse. A fragment of 4.0 kb had the highest specific radioactivity in terminally labelled DNA from several strains. One of these strains lacked the 120 kb prophage of SP beta that is normally integrated close to the terminus. Loss of the 120 kb prophage did not affect the point of termination which must therefore be regarded as a specific 'stop' sequence. Labelled terminus DNA has been used to identify lambda (Charon 4A) clones containing sequences derived from the terminal region. The total length of the restriction fragments present was 150 kb and adds another 90 kb to the 150 kb region mapped previously. Only one group of these sequences was present in a B. subtilis strain (CU1695) carrying a deletion spanning from SP beta to the right of the terminus and gltA. This suggests that the terminator sequence found in the wild-type can be deleted but presumably this strain has an alternative mechanism of termination.

Bacillus subtilis↗
PubMed1983

Identification of specific restriction fragments associated with a membrane subparticle from Bacillus subtilis.

When lysates of Bacillus subtilis were treated with restriction endonucleases EcoRI or HindIII, almost all of the DNA was released from the major plasma membrane fraction that was sedimentable at low speed. However, a very small part of the released DNA, when centrifuged at high speed, appeared to be bound to small membrane fragments. On agarose gels, this material, prepared with either enzyme, contained only a small number of restriction fragments, and the DNA in the sample hybridized with 11 to 12 EcoRI or HindIII fragments of chromosomal DNA. This DNA was used after nick-translation to screen Charon 4A clone banks for phages containing membrane-bound fragments. One of these was studied in detail. Only a part (about 5 kilobases) of the region present in this clone is important in binding the DNA to the membrane subparticle.

Bacillus subtilis↗
PubMed1982

Attachment of the chromosomal terminus of Bacillus subtilis to a fast-sedimenting particle.

After gently lysed protoplasts of exponential phase cells of Bacillus subtilis were treated with restriction endonuclease BamHI, 99% of the DNA did not sediment with the plasma membrane. This DNA was fractionated on sucrose gradients into (i) a fast-sedimenting fraction highly enriched for genes from the origin and terminus (purA and ilvA), (ii) a 50 to 100S component also enriched for purA and ilvA, and (iii) the bulk of the DNA. The fast-sedimenting fraction was dissociated by Sarkosyl; this fraction contained a substantial amount of protein and is probably a membrane subparticle. The S value of the 50 to 100S component was not greatly affected by Sarkosyl treatment, but these particles were unable to penetrate an agarose gel during electrophoresis and were retained by nitrocellulose filters. The terminus DNA in the fast-sedimenting fraction and the 50 to 100S component contained a large restriction fragment (1.5 x 10(7) to 2.0 x 10(7) daltons) encoding ilvA, thyB, and ilvD. The bulk of the SP beta prophage and metB, which lie to the right and left, respectively, of the ilvA-ilvD cluster, were not part of the complex. citK, which lies to the right of SP beta, appeared to be present in the fast-sedimenting complexes. The neighboring genes kauA and gltA were not part of the fast-sedimenting complexes. The presence of terminus DNA in the fast-sedimenting components was also demonstrated by a radiochemical method.

Bacillus subtilis↗
PubMed1980

Chromosome replication in sporulating cells of Bacillus subtilis.

A method of specifically labeling the chromosomal terminus of Bacillus subtilis is described. When sporulating cultures were pulse-labeled with [(3)H]thymidine and then treated with 6-(p-hydroxyphenylazo)uracil, a drug which inhibits deoxyribonucleic acid (DNA) synthesis rapidly and completely, the only labeled spores formed were those that had completed replication during the pulse period. DNA-mediated transformation was used to show that the DNA of spores formed in the presence of 6-(p-hydroxyphenylazo)uracil had the same ratio of origin to terminus markers as DNA from untreated spores. Furthermore, spores formed in the presence of 6-(p-hydroxyphenylazo)uracil had the same DNA content as untreated spores. These two observations indicated that spores formed in the presence of 6-(hydroxyphenylazo)uracil contained completed chromosomes. The rate of termination of chromosomes destined to be packaged into spores was determined by this method, using the Sterlini-Mandelstam replacement system and a single medium exhaustion system for inducing sporulation. With both systems the rate of termination reached a broad peak 2 h after the start of sporogenesis. This was measured from the time of resuspension by using the replacement system and from the point where exponential growth ceased in the exhaustion system. The amount of spore DNA synthesized in the Sterlini-Mandelstam sporulation-inducing medium was very close to one-half the amount of the DNA present in mature spores. This suggests that chromosomes destined to be packaged into spores were replicated from close to the origin and possibly initiated in the sporulation-inducing medium. A method was devised for estimating the time taken to complete replication of the chromosomes destined to be packaged into spores. This was probably no more than 50 min. Whereas starvation must have occurred almost simultaneously in most cells in the population, the chromosome replication that was essential for sporogenesis was distributed over a wide time span. Thus, in some cells, replication started within 10 min of the nutritional step-down, but the peak rate was not reached for 1 h; thereafter replication continued at a substantial rate.

Bacillus subtilis↗
PubMed1980

Specific labeling of the Bacillus subtilis chromosome terminus.

The deoxyribonucleic acid labeled by a procedure described previously for labeling the chromosomal terminus of B. subtilis 168 was substantially enriched for sequences homologous to bacteriophages SP beta and phi 3T, which integrate in the terminal region.

Bacillus subtilis↗