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Biomedical subjects

W Gilbert

Publications and source records attributed to W Gilbert.

At least 19 recordsLinked to original sources
PubMed1992

Novel DNA superstructures formed by telomere-like oligomers.

DNA oligomers containing three or more contiguous guanines form tetrastranded parallel complexes, G4-DNA, in the presence of alkali cations. However, oligomers that have a single multi-guanine motif at their 3' or 5' end, with a guanine as the terminal base, also form higher order products. Thus, the oligomer T8G3T forms a unique G4-DNA product at neutral pH in the presence of Na+, K+, or Rb+; however, its isomeric counterpart T9G3 in K+ or Rb+ generates an additional ladder of products of substantially lower gel mobility. We show that these larger complexes contain, respectively, 8, 12, or 16 distinct strands of oligomer. The octamer structure formed by T9G3 assembles in moderate salt at room temperature and melts around 60 degrees C in 100 mM KCl. Methylation protection experiments suggest a nested head-to-tail superstructure containing two tetraplexes bonded front-to-back via G quartets formed by out-of-register guanines. Naturally occurring chromosomal telomeres, which all have guanines at their 3' termini, may be able to form these superstructures.

Base Sequence↗
PubMed1991

DNA sequencing, today and tomorrow.

When we have the completed sequence, by about the year 2000, it will be possible to search more efficiently for genes involved in multifactorial diseases, including various cancers. With the genome sequenced and the full complement of genes identified, basic questions about our common biology will become central issues in the twenty-first century.

Base Sequence↗
PubMed1991

Neuronal differentiation and maturation in the mouse trigeminal sensory system, in vivo and in vitro.

We have isolated and characterized four monoclonal antibodies (mAbs B33, E1.9, B30, and B10) that recognize mouse trigeminal sensory neurons at specific times during development. These antibodies permit the study of neuronal differentiation, axon outgrowth, and neuronal maturation in the trigeminal sensory system. With B33, we can follow migrating neural crest and placode cells into the anlagen of the trigeminal ganglion. E1.9 immunoreactivity marks neuronal differentiation and appears in the central nervous system at embryonic day 8.5 (E8.5) and in the peripheral nervous system at E9, E1.9 and B30 show the axonal outgrowth of trigeminal sensory neurons and reveal the pioneering of the peripheral tracts by an early population of ganglionic neurons. At this stage, in the central nervous system, mesencephalic trigeminal neurons are also E1.9 and B30 positive as they migrate to their final location in the rostral metencephalon. B30 and B10 allow us to follow the maturation of these neurons. Also, in about 1% of the embryos, we identified mispositioned or misrouted trigeminal neurons. Furthermore, these biochemical markers facilitate the study of neuronal development in vitro. We find that, based on morphological and biochemical criteria, the maturation of trigeminal neurons in culture is target independent.

Animals↗
PubMed1991

The B30 ganglioside is a cell surface marker for neural crest-derived neurons in the developing mouse.

We have previously reported the isolation of a monoclonal antibody, mAb B30, that recognizes two minor gangliosides specifically expressed in a small subset of neurons in the developing mouse central nervous system (Stainier and Gilbert, 1989). B30 labels mesencephalic trigeminal neurons shortly after differentiation until about 2 weeks after birth. Postnatally, it also labels two specific monolayers of cerebellar neurons. In this study, we have characterized the B30 immunoreactivity in the developing peripheral nervous system of the mouse. We report that B30 is a marker for neural crest-derived neurons and have used it to follow the neuronal differentiation of neural crest cells in a serum-free chemically defined culture system. Within hours after plating, neural crest cells migrate away from the neural tube explant on a fibronectin or laminin substrate and by 24 hr, up to 15% of them have differentiated into morphologically identifable neurons. In vitro as in vivo, undifferentiated mouse neural crest cells express the GD3 ganglioside which is recognized by mAb B33, and neural crest-derived neurons can be labeled by mAbs B33, B30, and also E1.9, a specific neuronal cytoskeletal marker. We also show the unique biochemical specificity of mAb B30 and provide experimental evidence for the role of the B30 ganglioside in the cellular adhesion process.

Animals↗
PubMed1991

Zebrafish embryology and neural development.

The zebrafish is rapidly increasing in popularity with developmental biologists. Driving this interest are the elegant methods for in vivo observations and recovery of early developmental mutations. The past year has seen the introduction of additional methods for in vivo manipulation of identified cells and the application of these methods to mutant analysis.

Animals↗
PubMed1991

Spatial domains in the developing forebrain: developmental regulation of a restricted cell surface protein.

We have isolated a monoclonal antibody, mAb 52G9, that recognizes a 55-kDa cell surface protein restricted to the early embryonic rat forebrain and to placode-derived structures. In the central nervous system (CNS), 52G9 immunoreactivity appears at Embryonic Day 11 (E11) in the rostral-most area of the telencephalon. It then spreads to the neuroepithelium of the telencephalon and basal diencephalon. Most strikingly, it appears at E14 in a distinct zone at the caudal end of the ventral diencephalic neuroepithelium. This area is sharply defined by strong 52G9 immunoreactivity bounded by unlabeled neuroepithelium. The pattern revealed by 52G9 is the first biochemical demonstration of spatial domains in the forebrain at a time prior to neuronal differentiation. By E18, 52G9 immunoreactivity has progressively disappeared from the forebrain; the glomerular layer of the olfactory bulb is the only 52G9-positive area in the CNS. The olfactory, otic, and hypophyseal placodes, which can be identified as early as E10, are also 52G9 positive as are their derivatives, the sensory epithelial of the nasal passage and inner ear, and also Rathke's pouch. The distribution and regulation of the 52G9 protein suggests that this novel cell surface molecule may be involved in the formation of spatial domains in the developing forebrain.

Animals↗
PubMed1991

The limited universe of exons.

The catalogue of mosaic proteins showing evidence of exon-shuffling continues to expand. The repeated use of exon modules suggests that current protein diversity could have been generated from a finite set of such exon modules, and that the size and character of this underlying exon universe can still be glimpsed in extant proteins.

Animals↗
PubMed1991

Murine memory B cells are multi-isotype expressors.

Flow cytometric analyses of the surface immunoglobulins of murine memory B cells revealed the existence of populations expressing multiple isotypes, including an IgM+/IgG+ population that could be stimulated in vitro with antigen to secrete both IgM and IgG. Female BALB/c mice were immunized with R-phycoerythrin (RPE), a fluorescent photosynthetic accessory protein from red algae. Pooled splenocytes from these mice at different stages of immunization were stained with RPE as well as with allophycocyanin- and fluorescein-conjugated anti-isotype antibodies and analysed on a two-laser FACS. RPE-binding cell sub-populations were defined and selectively sorted to verify their phenotype and to demonstrate that the various subpopulations (IgM+/IgG+, IgM+/IgG-, IgM-/IgG+) had different isotype-secretion patterns when challenged with RPE in vitro. These results re-affirm the notion that a transcriptional processing mechanism may be responsible for the simultaneous expression of multiple isotypes in memory cells.

Animals↗
PubMed1990

How big is the universe of exons?

If genes have been assembled from exon subunits, the frequency with which exons are reused leads to an estimate of the size of the underlying exon universe. An exon database was constructed from available protein sequences, and homologous exons were identified on the basis of amino acid identity; statistically significant matches were determined by Monte Carlo methods. It is estimated that only 1000 to 7000 exons were needed to construct all proteins.

Amino Acid Sequence↗
PubMed1990

A sodium-potassium switch in the formation of four-stranded G4-DNA.

Single-stranded complex guanine-rich DNA sequences from chromosomal telomeres and elsewhere can associate to form stable parallel four-stranded structures termed G4-DNA by a process that is anomalously dependent on the particular alkali metal cation that is present. The anomaly, which is not found in the formation of G4-DNA by oligonucleotides containing short, single runs of three or more guanines, is caused by potassium cations excessively stabilizing fold-back intermediate structures, or pathway by-products.

Animals↗
PubMed1990

Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization.

We report a general method for the detection of restriction fragment length alterations associated with mutations or polymorphisms using whole genomic DNA rather than specific cloned DNA probes. We utilized a modified Southern Cross hybridization to display the hybridization pattern of all size-separated restriction fragments from wild-type Caenorhabditis elegans to all the corresponding fragments in a particular mutant strain and in a distinct C. elegans variety. In this analysis, almost all homologous restriction fragments are the same size in both strains and result in an intense diagonal of hybridization, whereas homologous fragments that differ in size between the two strains generate an off-diagonal spot. To attenuate the contribution of repeated sequences in the genome to spurious off-diagonal spots, restriction fragments from each genome were partially resected with a 3' or 5' exonuclease and not denatured, so that only the DNA sequences at the ends of these fragments could hybridize. Off-diagonal hybridization spots were detected at the expected locations when genomic DNA from wild-type was compared to an unc-54 mutant strain containing a 1.5 kb deletion or to a C. elegans variety that contains dispersed transposon insertions. We suggest that this modified Southern Cross hybridization technique could be used to identify restriction fragment length alterations associated with mutations or genome rearrangements in organisms with DNA complexities as large as 10(8) base pairs and, using rare-cutting enzymes and pulse-field gel electrophoresis, perhaps as large as mammalian genomes. This information could be used to clone fragments associated with such DNA alterations.

Animals↗
PubMed1990

Localization of an acetylcholine receptor intron to the nuclear membrane.

The first intron of the RNA for the acetylcholine receptor (AChR) alpha subunit shows a ringlike distribution around nuclei in multinucleated myotubes by in situ hybridization. This pattern is not observed for an actin intron or U1 RNA. Quantitation of the intron sequences reveals large variations in the amount of both the AChR and actin introns between nuclei within the same myotube, although all nuclei express equivalent amounts of U1 RNA. This differential RNA expression indicates that nuclei can individually control expression of messenger RNAs. The restricted distribution of the AChR intron RNA suggests a previously unknown step in RNA processing.

Actins↗