PubMed Health⌕ Search

Biomedical subjects

I A Hope

Publications and source records attributed to I A Hope.

At least 19 recordsLinked to original sources
PubMed1999

Complexity of developmental control: analysis of embryonic cell lineage specification in Caenorhabditis elegans using pes-1 as an early marker.

In the early Caenorhabditis elegans embryo five somatic founder cells are born during the first cleavages. The first of these founder cells, named AB, gives rise to 389 of the 558 nuclei present in the hatching larva. Very few genes directly involved in the specification of the AB lineage have been identified so far. Here we describe a screen of a large collection of maternal-effect embryonic lethal mutations for their effect on the early expression of a pes-1::lacZ fusion gene. This fusion gene is expressed in a characteristic pattern in 14 of the 32 AB descendants present shortly after the initiation of gastrulation. Of the 37 mutations in 36 genes suspected to be required specifically during development, 12 alter the expression of the pes-1::lacZ marker construct. The gene expression pattern alterations are of four types: reduction of expression, variable expression, ectopic expression in addition to the normal pattern, and reduction of the normal pattern together with ectopic expression. We estimate that approximately 100 maternal functions are required to establish the pes-1 expression pattern in the early embryo.

Animals↗
PubMed1998

Promoter trapping identifies real genes in C. elegans.

Promoter trapping involved screening uncharacterized fragments of C. elegans genomic DNA for C. elegans promoter activity. By sequencing the ends of these DNA fragments and locating their genomic origin using the available genome sequence data, promoter trapping has now been shown to identify real promoters of real genes, exactly as anticipated. Developmental expression patterns have thereby been linked to gene sequence, allowing further inferences on gene function to be drawn. Some expression patterns generated by promoter trapping include subcellular details. Localization to the surface of particular cells or even particular aspects of the cell surface was found to be consistent with the genes, now associated with these patterns, encoding membrane-spanning proteins. Data on gene expression patterns are easier to generate and characterize than mutant phenotypes and may provide the best means of interpreting the large quantity of sequence data currently being generated in genome projects.

Animals↗
PubMed1995

Developmental expression pattern screen for genes predicted in the C. elegans genome sequencing project.

Maximum use should be made of information generated in the genome sequencing projects. Toward this end, we have initiated a genome sequence-based, expression pattern screen of genes predicted from the Caenorhabditis elegans genome sequence data. We examined beta-galactosidase expression patterns in C. elegans lines transformed with lacZ reporter gene fusions constructed using predicted C. elegans gene promoter regions. Of the predicted genes in the cosmids analysed so far, 67% are amenable to the approach and 54% of examined genes yielded a developmental expression pattern. Expression pattern information is being made generally available using computer databases.

Animals↗
PubMed1994

PES-1 is expressed during early embryogenesis in Caenorhabditis elegans and has homology to the fork head family of transcription factors.

Promoter trapping has identified a gene, pes-1, which is expressed during C. elegans embryogenesis. The beta-galactosidase expression pattern, directed by the pes-1/lacZ fusion through which this gene was cloned, has been determined precisely in terms of the embryonic cell lineage and has three components. One component is in a subset of cells of the AB founder cell lineage during early embryogenesis, suggesting pes-1 may be regulated both by cell autonomous determinants and by intercellular signals. Analysis of cDNA suggests pes-1 has two sites for initiation of transcription and the two transcripts would encode related but distinct proteins. The predicted PES-1 proteins have homology to the fork head family of transcription factors and therefore may have important regulatory roles in early embryogenesis.

Amino Acid Sequence↗
PubMed1993

Molecular markers of differentiation in Caenorhabditis elegans obtained by promoter trapping.

Differentiation of specific cell types during animal development can be detected by monitoring expression of appropriate genes. For this study, six different beta-galactosidase expression patterns which can be used as differentiation markers in the nematode Caenorhabditis elegans are described. An earlier promoter trap screen identified pools of recombinant plasmids which gave patterns of beta-galactosidase expression when used to transform C. elegans. Each recombinant plasmid contained a random fragment of C. elegans genomic DNA fused upstream of a promoterless lacZ gene. Six of these pools were chosen, and individual pattern-producing plasmids within these pools were identified. The expression patterns have been characterized more thoroughly than in the original screen, thereby providing molecular markers for differentiation of several cell types. Many of the expression patterns involve more than one cell type. The genomic origin of the inserts of active plasmids were determined through localization on the physical genome map.

Animals↗
PubMed1991

'Promoter trapping' in Caenorhabditis elegans.

A screen of gene expression patterns has been developed for the nematode Caenorhabditis elegans. Promoter-reporter gene fusions were constructed in vitro by ligating C. elegans genomic DNA fragments upstream of a lacZ gene. Patterns of beta-galactosidase expression were examined by histochemical staining of C. elegans lines transformed with the constructs. beta-galactosidase expression depended on translational fusion, so constructs were assayed in large pools to expedite detection of the low proportion that were active. Expression in a variety of cell types and temporal patterns was observed with different construct pools. The most striking expression patterns were obtained when the beta-galactosidase activity was localized to subcellular structures by the C. elegans portion of the fusion protein. The active constructs of three selected pools were identified subsequently by an efficient combinatorial procedure. The genomic locations of the DNA fragments from the active constructs were determined and appear to define previously uncharacterized genetic loci.

Animals↗
PubMed1988

Structural and functional characterization of the short acidic transcriptional activation region of yeast GCN4 protein.

Derivatives of the yeast GCN4 transcription factor containing acidic regions of 35 to 40 amino acids fused directly to the DNA-binding domain are fully functional in vivo. High resolution deletion analysis and proteolytic mapping suggest that the activation region is a repeated structure composed of small units acting additively. Acidic character is a feature of the structural motif, possibly a dimer of alpha-helices from two GCN4 monomers, that may be important for interactions with the basic transcriptional machinery.

Amino Acid Sequence↗
PubMed1987

GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA.

The eukaryotic transcriptional activator protein, GCN4, synthesized in vitro from the cloned gene, binds specifically to the promoters of yeast amino acid biosynthetic genes. Previous analysis of truncated GCN4 derivatives localized the DNA binding domain to the C-terminal 60 amino acids and revealed that the size of the GCN4 derivative and the electrophoretic mobility of the protein-DNA complex were inversely related. This observation was utilized here to develop a novel method for determining the subunit structure of DNA binding proteins. A mixture of wild-type GCN4 protein and a smaller GCN4 derivative generated three complexes with DNA, two corresponding to those observed when the proteins are present individually and one new complex of intermediate mobility. This extra complex results from the heterodimer of the two GCN4 proteins of different sizes, demonstrating that GCN4 binds DNA as a dimer. The contacts sufficient for dimerization were localized to the 60 C-terminal amino acid, DNA binding domain, suggesting that dimerization of GCN4 is a critical aspect of specific DNA binding. Furthermore, stable GCN4 dimers were formed in the absence of target DNA. These observations suggest a structural model of GCN4 protein in which a dimer binds to overlapping and non-identical half-sites, explaining why GCN4 recognition sites act bidirectionally in stimulating transcription.

Chromosome Deletion↗
PubMed1986

Saturation mutagenesis of the yeast his3 regulatory site: requirements for transcriptional induction and for binding by GCN4 activator protein.

Expression of the yeast his3 and other amino acid biosynthetic genes is induced during conditions of amino acid starvation. The coordination of this response is mediated by a positive regulatory protein called GCN4, which binds specifically to regulatory sites upstream of all coregulated genes and stimulates their transcription. The nucleotide sequence requirements of the his3 regulatory site were determined by analysis of numerous point mutations obtained by a novel method of cloning oligonucleotides. Almost all single base pair mutations within the nine base pair sequence ATGACTCTT significantly reduce his3 induction in vivo and GCN4 binding in vitro, whereas changes outside this region have minimal effects. One mutation, which generates a sequence that most closely resembles the consensus for 15 coregulated genes, increases both the level of induction and the affinity for GCN4 protein. The palindromic nature of the optimal sequence, ATGACTCAT, suggest that GCN4 protein binds as a dimer to adjacent half-sites that possibly overlap.

Base Sequence↗
PubMed1986

Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast.

Yeast GCN4 protein binds specifically to the promoters of amino acid biosynthetic genes and coordinately induces their transcription. Serially deleted GCN4 and hybrid LexA-GCN4 proteins were assayed for specific DNA binding activity in vitro, and for stimulation of transcription in vivo. The specific DNA binding activity resides in the 60 C-terminal amino acids, a basic region of GCN4. However, certain deletions containing the entire DNA binding region are unable to activate transcription and instead act as repressors in vivo. The activation function appears to critically involve just 19 amino acids that are centrally located in an acidic region of GCN4. In addition to their functional separation, the DNA binding and transcriptional activation regions of the protein can be separated physically by elastase cleavage. The implications of these results for the mechanisms of DNA sequence recognition and transcription activation are discussed.

Binding Sites↗
PubMed1985

The gene for an exported antigen of the malaria parasite Plasmodium falciparum cloned and expressed in Escherichia coli.

An exported protein of the erythrocytic stages of the malaria parasite, Plasmodium falciparum, has epitope(s) in common with the surface of the sporozoite stage (1). Two cDNA clones encoding this protein, Ag5.1, have now been isolated and expressed in Escherichia coli. The coding sequence contains a region with strong homology to that of the circumsporozoite protein of P. falciparum. Other features of the sequence can be explained in terms of the observed behaviour of the protein in the parasite life cycle. The Ag5.1 can now be synthesised in bacteria in sufficient amounts to analyse the immune response to this protein.

Amino Acid Sequence↗
PubMed1985

GCN4 protein, synthesized in vitro, binds HIS3 regulatory sequences: implications for general control of amino acid biosynthetic genes in yeast.

The yeast GCN4 gene product is necessary for the transcriptional induction of many amino acid biosynthetic genes in response to conditions of amino acid starvation. We synthesized radioactively pure GCN4 protein by in vitro translation of mRNA produced by in vitro transcription with SP6 RNA polymerase. GCN4 protein binds specifically to the 20 bp region of the HIS3 gene that is critical for transcriptional regulation in vivo and contains the TGACTC sequence common to coregulated genes. A synthetic GCN4 mutant protein lacking the 40 C-terminal amino acids fails to bind DNA; this correlates with a gcn4 mutant gene that is nonfunctional in vivo. Finally, GCN4 protein binds to the promoter regions of coordinately regulated genes, but not to analogous regions of other genes. We suggest that GCN4 protein is a specific transcription factor, and we describe a molecular model for the general control of amino acid biosynthetic genes.

Base Sequence↗
PubMed1984

Characterisation and translation studies of messenger RNA from the human malaria parasite Plasmodium falciparum and construction of a cDNA library.

RNA was isolated from trophozoites, schizonts and mixed populations of Plasmodium falciparum. 5% of the total was poly(A+) message, of average length 1.2 kb (10-12 kb maximum) and a poly(A) content of 10%. The mRNA fractions could be translated in vitro by reticulocyte lysates supplemented either with exogenous or P. falciparum tRNA. The patterns from two independent isolates, one cloned (T9-94) and one uncloned (K1) were virtually identical. Major translation products from 16-230 kDa have been measured. The most abundant is lactate dehydrogenase (34.8 kDa). Trophozoite mRNA codes principally for proteins of less than or equal to 93 kDa, while schizont mRNA codes for additional proteins of higher molecular mass. There are marked similarities between the in vitro translation products and proteins synthesised in vivo in synchronous cultures. A number of schizont mRNA translation products (principally those of 230, 203, 185, 170, 115, 101 and 71 kDa) are specifically precipitated without post-translational modification by sera from humans exposed to malaria. A cDNA library has been constructed in phage lambda from total poly(A+) RNA and partially characterised. About 10% of the clones derive from abundant mRNA sequences. Putative actin clones have been isolated from this library and the parasite actin mRNA sized at approx. 2.8 kb.

Actins↗