Search PubMed⌕ Search

Biomedical subjects

J D McGhee

Publications and source records attributed to J D McGhee.

At least 19 recordsLinked to original sources
PubMed1995

elt-2, a second GATA factor from the nematode Caenorhabditis elegans.

We have previously shown that a tandem pair of (A/T)GATA(A/G) sequences in the promoter region of the Caenorhabditis elegans gut esterase gene (ges-1) controls the tissue specificity of ges-1 expression in vivo. The ges-1 GATA region was used as a probe to screen a C. elegans cDNA expression library, and a gene for a new C. elegans GATA-factor (named elt-2) was isolated. The longest open reading frame in the elt-2 cDNA codes for a protein of M(r) 47,000 with a single zinc finger domain, similar (approximately 75% amino acid identity) to the C-terminal fingers of all other two-fingered GATA factors isolated to date. A similar degree of relatedness is found with the single-finger DNA binding domains of GATA factors identified in invertebrates. An upstream region in the ELT-2 protein with the sequence C-X2-C-X16-C-X2-C has some of the characteristics of a zinc finger domain but is highly diverged from the zinc finger domains of other GATA factors. The elt-2 gene is expressed as an SL1 trans-spliced message, which can be detected at all stages of development except oocytes; however, elt-2 message levels are 5-10-fold higher in embryos than in other stages. The genomic clone for elt-2 has been characterized and mapped near the center of the C. elegans X chromosome, ELT-2 protein, produced by in vitro transcription-translation, binds to ges-1 GATA-containing oligonucleotides similar to a factor previously identified in C. elegans embryo extracts, both as assayed by electrophoretic migration and by competition with wild type and mutant oligonucleotides. However, there is as yet no direct evidence that elt-2 does or does not control ges-1.

Amino Acid Sequence↗
PubMed1995

A gut-to-pharynx/tail switch in embryonic expression of the Caenorhabditis elegans ges-1 gene centers on two GATA sequences.

The Caenorhabditis elegans ges-1 gene (gut esterase No. 1) is expressed only in the intestinal lineage, beginning when the developing gut has only four to eight cells. We analyze the sequence requirements for this tissue-specific gene regulation by injecting deleted/mutated constructs of the ges-1 gene into a viable ges-1 (null) strain of worms and assaying heritably transformed embryos by esterase histochemistry. Many deletion constructs accurately reconstitute the wildtype gut-specific ges-1 expression. However, deletions in the neighborhood of 1100 bp upstream of the ges-1 ATG abolish ges-1 expression in the developing gut, while at the same time activating ges-1 expression in cells of the pharynx/tail that appear to belong to the sister lineage of the gut. Deletions of a 36-bp DNA region containing two tandem WGATAR sequences are sufficient to cause this gut-to-pharynx/tail switch in expression pattern. Deletion of either one of the WGATAR sites or deletion of an adjoining downstream region directs ges-1 expression only in a restricted set of cells of the anterior gut. The ges-1 GATA region acts like a gut-specific enhancer in that: (i) it restores ges-1 gut expression when reinserted elsewhere into the GATA-deleted ges-1 gene; and (ii) multiple copies direct gut expression of an hsp16-lacZ reporter gene. The ges-1 GATA-region also acts as the site of the pharynx/tail repression in that reinsertion elsewhere into the GATA-deleted ges-1 construct causes repression of ges-1 in the pharynx/tail. However, multiple copies of the GATA region are not able to repress the heat-induced expression of an hsp16-lacZ reporter gene, suggesting that the pharynx/tail repression mechanism is specific to the ges-1 environment. Finally, mutation rather than deletion of the individual GATA sequences suggests that gut activation and pharynx/tail repression may be due to separate factors. We present a molecular model that summarizes these results. The ges-1 control circuitry appears surprisingly complex for what might have been expected to be the simplest possible example of a nonessential gene expressed early in a clonal embryonic lineage.

Animals↗
PubMed1995

The C. elegans neuronally expressed homeobox gene ceh-10 is closely related to genes expressed in the vertebrate eye.

We describe the homeobox gene ceh-10 from the nematode Caenorhabditis elegans. The homeodomain of ceh-10 is closely related to the homeodomains of two genes recently cloned from the vertebrate retina, Chx10 from mice and Vsx-1 from goldfish. We show that the sequence conservation extends well beyond the homeodomain and includes a region (named the CVC domain) of roughly 60 amino acids immediately C-terminal to the homeodomain. As assayed in transgenic worms, the promoter region of ceh-10 directs expression of a lacZ reporter gene to a small number of neurons. We draw a parallel between the bipolar cells of the inner nuclear layer of the vertebrate retina, which express Chx10 and Vsx-1, and an interneuron in C. elegans called AIY, which expresses ceh-10. AIY receives synaptic input from a sensory cell, just as do bipolar cells of the vertebrate retina. In C. elegans, the sensory cell AFD is not known to be photosensitive but is known to be thermosensitive; moreover, a cell with similar position in the amphids of other nematodes has been suggested indeed to be photosensitive. Our results emphasize the highly conserved nature of sensory regulatory mechanisms and suggest one way in which photosensitive organelles might have originated in evolution.

Amino Acid Sequence↗
PubMed1992

DNA synthesis in the early embryo of the nematode Ascaris suum.

We have used microspectrofluorimetry to measure the rate of DNA synthesis in the first two embryonic cell cycles of the parasitic nematode Ascaris suum. The S-phase of the early Ascaris cell cycles occupies at most 1 hr; G2 phase is prominent and occupies approximately 11 hr; no G1 phase could be detected. These results contrast with our previous measurements made with embryos of the free-living nematode Caenorhabditis elegans, in which the earliest cell cycles consist of simple alternations between S and M phases.

Animals↗
PubMed1991

Resolution of sequencing ambiguities: a universal FokI adapter permits Maxam-Gilbert re-sequencing of single-stranded phagemid DNA.

We propose a method to resolve ambiguities encountered when single-stranded (ss) phagemid DNA templates are sequenced by the dideoxy method. A single oligodeoxyribonucleotide (oligo) is synthesized with the following features: (i) the 20 nucleotides (nt) at the 5'-end form a double-stranded hairpin containing a FokI restriction site, exactly as previously described by Podhajska and Szybalski [Gene 40 (1985) 175-182]; (ii) the 23 nt at the 3'-end hybridize to the (+)strand of ss phagemid DNA in the region complementary to the M13 universal sequencing primer. In a simple one-tube set of reactions, ss phagemid DNA is annealed to this oligo, cleaved by FokI at a unique site outside the vector multiple cloning site and then labelled at this unique site by Klenow polymerase and [alpha-32P]dCTP. These reactions provide a convenient route by which Maxam-Gilbert chemical degradation sequencing methods can be used to resolve ambiguities encountered in the dideoxy-sequencing of a unidirectional deletion series already prepared in popular phagemid vectors. A single oligo allows labelling of all members of a deletion series. A second universal oligo allows the same set of reactions to be applied to inserts cloned into (-)strand phagemids.

Animals↗
PubMed1991

Spatial control of gut-specific gene expression during Caenorhabditis elegans development.

The nematode Caenorhabditis elegans was transformed with constructs containing upstream deletions of the gut-specific ges-1 carboxylesterase gene. With particular deletions, ges-1 was expressed, not as normally in the gut, but rather in muscle cells of the pharynx (which belong to a sister lineage of the gut) or in body wall muscle and hypodermal cells (which belong to a cousin lineage of the gut). These observations suggest that gut-specific gene expression in C. elegans involves not only gut-specific activators but also multiple repressors that are present in particular nongut lineages.

Animals↗
PubMed1991

An acid phosphatase as a biochemical marker for intestinal development in the nematode Caenorhabditis elegans.

We describe an acid phosphatase enzyme (EC 3.1.3.2) that is localized to the intestine of the nematode Caenorhabditis elegans and that should serve as a convenient biochemical marker for gut differentiation. In adult worms, acid phosphatase activity is located along the edge of the gut lumen in the vicinity of the intestinal brush border. All but the anterior six cells of the intestine stain for phosphatase activity; the nonstaining cells all descend from the Ea(l/r)(a/p)a cells. Acid phosphatase activity is low in oocytes and early embryos but increases substantially when embryos reach late morphogenesis stage; this increase corresponds to the appearance of a major band of acid phosphatase activity detectable on isoelectric focusing gels. We designate this band as the product of the pho-1 gene. The pattern of acid phosphatase expression in several embryonic mutants suggests that pho-1 expression in the developing intestine is lineage autonomous. We induced an isoelectric focusing variant in the pho-1 enzyme and used this to map the pho-1 locus about 1.5 map units to the left of center of chromosome II. We purified the pho-1 enzyme to homogeneity (6500-fold purification; 4% recovery of activity); the pho-1 acid phosphatase is a homodimeric glycoprotein with a subunit molecular weight of 55,000 Da. This paper establishes a new experimental system with which to investigate the molecular basis of lineage-specific gene expression during C. elegans development.

Acid Phosphatase↗
PubMed1990

Homeobox containing genes in the nematode Caenorhabditis elegans.

We designed a unique 36-mer oligonucleotide probe, based on the most highly conserved amino acid sequences of Antennapedia-like homeodomains and the codon bias of Caenorhabditis elegans. This probe was then used to isolate four classes of genes from a C. elegans genomic library. Sequencing reveals that we have isolated three new homeobox genes, designated ceh-1, ceh-9 and ceh-10. The fourth homeobox gene, ceh-11, has recently been described by Schaller et al (Nucleic Acids Res. 18, 2033-2036). The amino acid sequence of ceh-1 is 87% similar to the honeybee H40 homeodomain, 85% similar to the Drosophila NK-1 homeodomain and 82% similar to the chicken CHox3 homeodomain. The sequence ceh-10 appears to be a member of the paired class of homeodomains. The other two sequences, ceh-9 and ceh-11, remain unclassified. Three of the four sequences have at least one intron within the homeobox region. Transcripts of ceh-10 and ceh-11 are present in embryonic RNA but are greatly diminished in later developmental stages. Three of the four new genes have been placed on the C. elegans genomic map.

Amino Acid Sequence↗
PubMed1990

Are many Z-DNA binding proteins actually phospholipid-binding proteins?

We used a Z-DNA affinity column to isolate a collection of Z-DNA binding proteins from a high salt extract of Escherichia coli. We identified one of the major Z-DNA binding proteins of this fraction, not as a protein involved in gene regulation or genetic recombination, but rather as an outer membrane porin protein. We then showed that several other known phospholipid-binding proteins (bovine lung annexins and human serum lipoproteins) also bind much more tightly to Z-DNA than to B-DNA. In all cases, this Z-DNA binding was strongly blocked by competition with acidic phospholipids, such as cardiolipin. Our results raise the question whether many of the Z-DNA binding proteins previously isolated are actually phospholipid-binding proteins.

Carrier Proteins↗
PubMed1990

Production of null mutants in the major intestinal esterase gene (ges-1) of the nematode Caenorhabditis elegans.

The ges-1 gene of the nematode Caenorhabditis elegans codes for a nonspecific carboxylesterase that is expressed only in the intestinal lineage. This esterase has turned out to be a convenient biochemical marker for lineage-specific differentiation. In the present paper, we describe the production of several C. elegans strains that lack detectable activity of the ges-1 esterase. To isolate these ges-1 null strains, we first produced a strain of hermaphrodites in which the wild-type copy of the ges-1 gene was stably balanced over a previously isolated isoelectric focusing allele, ges-1(ca6); this parental strain was then mutagenized with EMS and isoelectric focusing gels were used to identify progeny populations that lacked either ges-1(+) or ges-1(ca6) esterase activity. This method is a straightforward and general approach to obtaining null mutations in any gene that has a biochemical or immunological assay. The ges-1 gene is not essential to worm survival, development or reproduction. Furthermore, lack of the ges-1 product has no obvious effect on the ability of worms (containing either normal or greatly reduced levels of acetylcholinesterases) to survive exposure to esterase inhibitors. The ges-1 gene product provides roughly half of the total esterase activity measured in crude extracts of L1 larvae or mixed worm populations. However, histochemical staining of individual ges-1(0) embryos shows that the ges-1 esterase is the first and essentially the only esterase to be produced during embryonic development, from the midproliferation phase up to at least the twofold stage of morphogenesis. These ges-1(0) strains now allow us to investigate the developmental control of the ges-1 gene by DNA-mediated transformation, in which the ges-1 gene acts as its own reporter.

Animals↗
PubMed1988

Yolk proteins from nematodes, chickens, and frogs bind strongly and preferentially to left-handed Z-DNA.

Yolk proteins purified from the nematode Caenorhabditis elegans, from the frog Xenopus laevis, and from chicken eggs all have the unexpected property of binding strongly and preferentially to a left-handed Z-DNA probe, brominated poly(dG-dC). We estimate that the nematode proteins bind to Z-DNA with an association constant of at least 10(4) (M-1) and that this association constant is at least 40-50-fold higher than the association constant to B-DNA. Thus, yolk proteins have a higher Z-DNA specificity than most of the Z-DNA binding proteins previously isolated from other sources. Although yolk protein binding to Z-DNA is poorly competed by a wide variety of nucleic acids, the interaction is strongly competed by the phospholipids cardiolipin and phosphatidic acid (500-1000-fold better than by the same mass of B-DNA). We suggest that Z-DNA interacts with the yolk protein phospholipid binding site. In general, our results emphasize the danger of using physical properties to infer biological function. In particular, our results should raise serious questions about the biological relevance of previously isolated Z-DNA binding proteins.

Animals↗
PubMed1988

DNA synthesis and the control of embryonic gene expression in C. elegans.

DNA synthesis in each cell lineage of the early C. elegans embryo was measured using microspectrofluorimetry. Aphidicolin was shown to inhibit DNA synthesis almost instantly and completely. Aphidicolin was then used to investigate how DNA synthesis controls expression of two biochemical markers that appear at different times during gut development: gut granules and a carboxylesterase. We show that marker expression is controlled neither by reaching the normal DNA: cytoplasm ratio, by counting the normal number of rounds of DNA synthesis, nor by a simple lengthening of the cell cycle. Instead, expression of both gut markers requires a short period of DNA synthesis in the first cell cycle after the gut has been clonally established.

Amanitins↗
PubMed1988

Paternal DNA strands segregate to both trophectoderm and inner cell mass of the developing mouse embryo.

The localization of sperm DNA strands was examined in preimplantation mouse embryos. Male mice were treated with bromodeoxyuridine (BrdU) to label germ-cell-line DNA and were then mated with unlabeled females. Sperm DNA strands in early embryos derived from these matings could be detected by means of a fluorescent antibody specific to BrdU. The position and number of fluorescent spots detected in the developing embryos are consistent with a model in which paternal DNA strands segregate at random into both the trophectoderm and the inner cell mass. Although we could not follow the segregation of individual paternal chromosomes, we could detect no overall segregation pattern of the sperm DNA strands that could be obviously related to chromosome imprinting.

Animals↗
PubMed1987

Purification and characterization of a carboxylesterase from the intestine of the nematode Caenorhabditis elegans.

The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N-terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].

Amino Acid Sequence↗
PubMed1987

Parental DNA strands segregate randomly during embryonic development of Caenorhabditis elegans.

The fate of gamete DNA was followed in the next generation embryos of the nematode C. elegans. Either male worms or spermless hermaphrodites were grown on bromodeoxyuridine-containing E. coli in order to label germ-line DNA. Matings then produced embryos in which only the DNA strands provided by the gametes contained label. This original gamete DNA could be detected during embryonic development by using a fluorescently labeled monoclonal antibody specific to bromodeoxyuridine. Both the number and position of fluorescent spots in the embryo indicate that gamete DNA strands segregate randomly during development. Random segregation of parental DNA strands rules out models of development that invoke chromosome imprinting or immortal DNA strands.

Animals↗