Biomedical subjects
K Moses
Publications and source records attributed to K Moses.
Identification and functional characterization of conserved promoter elements from glass: a retinal development gene of Drosophila.
The Drosophila melanogaster glass transcription factor acts in photoreceptor cell development, glass transcription in the developing eye begins in the morphogenetic furrow. We report a deletion analysis which shows that a 5147 bp fragment can confer near wild-type expression in the developing eye. We completed the sequence of a similar Drosophila virilis fragment and comparison of these two genomic sequences reveals seven perfectly conserved sequence elements of 15 bp (or longer). We placed oligomers of each element 5' to an hsp70 promoter-lacZ fusion construct, and examined the patterns of beta-galactosidase expression produced in transgenic files. Three of these elements drive beta-galactosidase expression in the developing eye, and one is inhibitory.
Identification and characterization of autosomal genes that interact with glass in the developing Drosophila eye.
The glass gene encodes a zinc finger, DNA-binding protein that is required for photoreceptor cell development in Drosophila melanogaster. In the developing compound eye, glass function is regulated at two points: (1) the protein is expressed in all cells' nuclei posterior to the morphogenetic furrow and (2) the ability of the Glass protein to regulate downstream genes is largely limited to the developing photoreceptor cells. We conducted a series of genetic screens for autosomal dominant second-site modifiers of the weal allele glass3, to discover genes with products that may regulate glass function at either of these levels. Seventy-six dominant enhancer mutations were recovered (and no dominant suppressors). Most of these dominant mutations are in essential genes and are associated with recessive lethality. We have assigned these mutations to 23 complementation groups that include multiple alleles of Star and hedgehog as well as single alleles of Delta, roughened eye, glass and hairy. Mutations in 18 of the complementation groups are embryonic lethals, and of these, 13 show abnormal adult retinal phenotypes in homozygous clones, usually with altered numbers of photoreceptor cells in some of the ommatidia.
Extracellular regulators and pattern formation in the developing Drosophila retina.
The compound eye of Drosophila melanogaster is composed of about 800 similar facets (or ommatidia) each of which contains 20 cells (8 photoreceptor neurons and 12 accessory cells). The fly's vision depends on the precise geometry of these components, and the developmental systems that produce pattern and cell fate have become models for nervous system development in general. The development of this pattern is progressive and appears to involve several levels of induction and inhibition, mediated by diffusible signals. The analysis of genes identified by mutation has revealed that several diffusible factors act in the early patterning steps of the eye. Genes that encode these factors include hedgehog, decapentaplegic, wingless, spitz and scabrous.
Mechanisms of Drosophila retinal morphogenesis: the virtues of being progressive.
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The product of hedgehog autoproteolytic cleavage active in local and long-range signalling.
The secreted protein products of the hedgehog (hh) gene family are associated with local and long-range signalling activities that are responsible for developmental patterning in multiple systems, including Drosophila embryonic and larval tissues and vertebrate neural tube, limbs and somites. In a process that is critical for full biological activity, the hedgehog protein (Hh) undergoes autoproteolysis to generate two biochemically distinct products, an 18K amino-terminal fragment, N, and a 25K carboxy-terminal fragment, C (ref. 16); mutations that block autoproteolysis impair Hh function. We have identified the site of autoproteolytic cleavage and find that it is broadly conserved throughout the hedgehog family. Knowing the site of cleavage, we were able to test the function of the N and C cleavage products in Drosophila assays. We show here that the N product is the active species in both local and long-range signalling. Consistent with this, all twelve mapped hedgehog mutations either affected the structure of the N product directly or otherwise blocked the release of N from the Hh precursor as a result of deletion or alteration of sequences in the C domain.
Wingless and patched are negative regulators of the morphogenetic furrow and can affect tissue polarity in the developing Drosophila compound eye.
In the developing Drosophila compound eye, a wave of pattern formation and cell-type determination sweeps across the presumptive eye epithelium. This 'morphogenetic furrow' coordinates the epithelial cells' division cycle, shape and gene expression to produce evenly spaced neural cell clusters that will eventually form the adult ommatidia. As these clusters develop, they rotate inwards to face the eye's equator and establish tissue polarity. We have found that wingless is strongly expressed in the dorsal margin of the presumptive eye field, ahead of the morphogenetic furrow. We have shown that inactivation of Wingless results in the induction of an ectopic furrow that proceeds ventrally from the dorsal margin. This ectopic furrow is normal in most respects, however the clusters formed by it fail to rotate, and we propose a two-vector model to account for normal rotation and tissue polarity in the retina. A second consequence of this inactivation of Wingless is that the dorsal head is largely deleted. We have also found that patched loss-of-function mosaic clones induce circular ectopic morphogenetic furrows (consistent with the observations of other workers with the hedgehog, and PKA genes). We use such patched induced furrows to test the two-vector model for cluster rotation and tissue polarity.
spitz, a Drosophila homolog of transforming growth factor-alpha, is required in the founding photoreceptor cells of the compound eye facets.
Cell type specification and differentiation in the developing Drosophila compound eye begins in the morphogenetic furrow. In the furrow, cells are organized into evenly spaced preclusters and there is a synchronized arrest of the cells' mitotic cycle in G1. We report that recessive spitz loss-of-function mutations affect compound eye development. Spitz is homologous to the human transforming growth factor-alpha. In mosaic clones, spitz function is required in the first photoreceptor cells to differentiate for normal ommatidial development. spitz loss-of-function mutations are dominant suppressors of EgfrE gain-of-function mutations of the epidermal growth factor-receptor gene. These data suggest that the spitz product is a precluster promoting factor. spitz transcription increases abruptly in the morphogenetic furrow, the obverse of Egfr expression. We present a model for the expression of, and cellular requirement for, this growth factor homolog.
A unique subpopulation of murine DNA polymerase alpha/primase specifically interacts with polyomavirus T antigen and stimulates DNA replication.
Murine cells or cell extracts support the replication of plasmids containing the replication origin (ori-DNA) of polyomavirus (Py) but not that of simian virus 40 (SV40), whereas human cells or cell extracts support the replication of SV40 ori-DNA but not that of Py ori-DNA. It was shown previously that fractions containing DNA polymerase alpha/primase from permissive cells allow viral ori-DNA replication to proceed in extracts of nonpermissive cells. To extend these observations, the binding of Py T antigen to both the permissive and nonpermissive DNA polymerase alpha/primase was examined. Py T antigen was retained by a murine DNA polymerase alpha/primase but not by a human DNA polymerase alpha/primase affinity column. Likewise, a Py T antigen affinity column retained DNA polymerase alpha/primase activity from murine cells but not from human cells. The murine fraction which bound to the Py T antigen column was able to stimulate Py ori-DNA replication in the nonpermissive extract. However, the DNA polymerase alpha/primase activity in this murine fraction constituted only a relatively small proportion (approximately 20 to 40%) of the total murine DNA polymerase alpha/primase that had been applied to the column. The DNA polymerase alpha/primase purified from the nonbound murine fraction, although far more replete in this activity, was incapable of supporting Py DNA replication. The two forms of murine DNA polymerase alpha/primase also differed in their interactions with Py T antigen. Our data thus demonstrate that there are two distinct populations of DNA polymerase alpha/primase in murine cells and that species-specific interactions between T antigen and DNA polymerases can be identified. They may also provide the basis for initiating a novel means of characterizing unique subpopulations of DNA polymerase alpha/primase.
The segment polarity gene hedgehog is required for progression of the morphogenetic furrow in the developing Drosophila eye.
Cell-type specification in the Drosophila compound eye begins at the morphogenetic furrow. The furrow sweeps across the developing eye epithelium and is coincident with four classes of cellular events: coordinated changes in cell shape, changes in gene expression, synchronization of the cell cycle, and the specification of a regular array of ommatidial founder cells. The molecular mechanisms that induce these events in the developing eye have hitherto been unknown. We identify here a gene specifically required for furrow progression, hedgehog (hh). We show that hh expression posterior to the morphogenetic furrow is continuously required for its progression. We propose that forward diffusion of hh protein induces anterior cells to enter the furrow.
Mutations on the second chromosome affecting the Drosophila eye.
In the developing eye of Drosophila, cell interactions appear to be responsible for organising undifferentiated cells into unit eyes, or ommatidia. Extensive mutagenesis has been used to search for mutations affecting the development and differentiation of ommatidia. These mutations have been characterized using sections of adults and immunocytochemistry of imaginal discs. Fourteen loci on the second chromosome are described that affect the spacing of the preclusters, the differentiation of ommatidial cells, orientation of the ommatidia, or architecture of the adult retina, that cause retinal degeneration in larval or pupal eye discs, or that cause homeotic transformation of part of the head.
The role of transcription factors in the developing Drosophila eye.
In the developing Drosophila compound eye, multipotent precursor cells are induced to develop into particular cell types through sequential induction. In the target cells, transcription factors may be modulated by the inductive signals to execute their instructions. Four recently isolated genes may encode such developmentally modulated transcription factors.
Glass encodes a site-specific DNA-binding protein that is regulated in response to positional signals in the developing Drosophila eye.
The glass gene encodes a zinc finger protein required for normal photoreceptor cell development in Drosophila. We show that glass transcripts are present in the third-instar eye-imaginal disc starting in the morphogenetic furrow and extending to the posterior margin of the disc; glass protein is detected in the nuclei of all cells in this region. We also show that glass encodes a site-specific DNA-binding protein. A 27-bp glass-binding site can confer glass-dependent expression on a reporter gene in developing photoreceptor cells, the particular subset of glass-expressing cells known to require glass function. This specificity may represent a regulation of glass protein activity after cells are recruited to the photoreceptor cell fate.
Development and testing of a carbohydrate monitoring tool for athletes.
Improving carbohydrate intake in athletes will increase muscle glycogen storage. This in turn can improve exercise time and performance by delaying fatigue. However, planning and consuming a diet that contains 60% to 70% carbohydrate is difficult for most athletes. To develop a simple carbohydrate monitoring tool for athletes, we analyzed three sets of 3-day diet records from 17 male endurance runners over a 10-week competitive period and 9 female endurance runners over a 9-week training period. We then developed a simple carbohydrate monitoring tool. To validate the instrument, we compared each athlete's carbohydrate intake as estimated using the instrument with the athlete's actual carbohydrate intake from the 3-day diet records. Mean estimated percents of energy from carbohydrate using the carbohydrate monitoring tool were not significantly different from the actual mean carbohydrate intakes. Examination of individual diets showed that the estimated carbohydrate was always within 2% of the actual carbohydrate. Therefore, the instrument did a good job of estimating the percent of energy from carbohydrate in the diets of endurance runners. This instrument will provide a quick method by which the athlete can assess and improve carbohydrate intake on a daily basis without the use of daily diet records.
Correlation of enhanced 6-mercaptopurine cytotoxicity with increased phosphoribosylpyrophosphate levels in Chinese hamster ovary cells treated with 3-aminobenzamide.
3-Aminobenzamide (3AB) has been used widely to inhibit the nuclear enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30) and study the involvement of poly(ADP-ribose) synthesis in DNA repair and other cellular functions. 3AB (3 mM) potentiates the cytotoxicity of 6-mercaptopurine (MP) and azathioprine in CHO-K1 cells with dose enhancement factors at 10% survival of 30-fold. In synchronized cells, 3AB is required during G1 and early S phase to obtain potentiation of MP cytotoxicity. There is a small but significant depletion of cellular NAD in MP-treated cells. As demonstrated by flow cytometric analysis, 20-40 microM MP causes an accumulation of cells in early S phase of the cell cycle. 3AB (3 mM) has no effect on cell cycle distribution; however, in the presence of MP, a similar accumulation is seen by 2-5 microM MP. 3AB and MP per se have no effect on phosphoribosylpyrophosphate levels, but coincubation causes a 30-fold increase in phosphoribosylpyrophosphate levels, reaching a maximum by 1.5 microM MP and declining to basal levels by 10 microM MP. There was a good correlation between the 3AB dose-dependent increase in cell killing and rise in phosphoribosylpyrophosphate levels.
The Adh gene promoters of Drosophila melanogaster and Drosophila orena are functionally conserved and share features of sequence structure and nuclease-protected sites.
The sibling species Drosophila melanogaster and D. orena show similar patterns of alcohol dehydrogenase expression, both spatially and temporally. These two species diverged from a common ancestor 6 million to 15 million years ago, and the DNA sequences of the promoter regions of their Adh genes show a mosaic pattern of conservation and change. By interspecific transformation of D. orena sequences into D. melanogaster, we demonstrate a functional equivalence between these sequences. Using both D. melanogaster embryo extracts and purified transcription factor Adf-1, we compare the protection of these promoter sequences from nuclease, demonstrating considerable conservation.
The glass gene encodes a zinc-finger protein required by Drosophila photoreceptor cells.
Null mutations of glass specifically remove photoreceptor cells, leaving other cell types intact. We have isolated the glass gene and have shown that its transcript encodes a putative protein of 604 amino acids with five zinc-fingers. The glass product may be a transcription factor required for the development of a single neuronal cell type.
Synergistic enhancement of 6-thioguanine cytotoxicity by ADP-ribosyltransferase inhibitors.
The effect of the adenosine diphosphoribosyltransferase inhibitors, the substituted benzamides, on the cytotoxicity of 6-thioguanine (6TG) was investigated. Nontoxic concentrations of benzamides potentiated the cytotoxicity of 6TG with a dose enhancement factor of 2, producing a 6-fold increase in cell killing at 10% survival. 6TG treatment did not deplete cellular NAD levels, and in the presence of 3-aminobenzamide, there was no increase in the number of 6TG-induced DNA strand breaks. To obtain potentiation of cytotoxicity, 3-aminobenzamide had to be present in late G1-S phase during the cell cycle in which 6TG is incorporated into the DNA. These data indicate that the substituted benzamides potentiate the cytotoxicity of 6TG by a mechanism independent of an inhibition of DNA repair.