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S B Carroll

Publications and source records attributed to S B Carroll.

At least 73 records · Page 4Linked to original sources
PubMed1989

The zygotic control of Drosophila pair-rule gene expression. I. A search for new pair-rule regulatory loci.

The examination of pair-rule gene expression in wild-type and segmentation mutant embryos has identified many, but not necessarily all, of the elements of the regulatory system that establish their periodic patterns. Here we have conducted a new type of search for previously unknown regulators of these genes by examining pair-rule gene expression in blastoderm embryos lacking parts of or entire chromosomes. This method has the advantage of direct inspection of abnormal pair-rule gene patterns without relying upon mutagenesis or interpretation of larval phenotypes for the identification of segmentation genes. From these experiments we conclude that: (i) most zygotically required regulators of the fushi tarazu (ftz), even-skipped (eve) and hairy (h) pair-rule genes have been identified, except for one or more loci we have uncovered on chromosome arm 2L; (ii) the repression of the ftz and eve genes in the anterior third of the embryo is under maternal, not zygotic control; and (iii) there are no general zygotically required activators of pair-rule gene expression. The results suggest that the molecular basis of pair-rule gene regulation can be pursued with greater confidence now that most key trans-acting factors are already in hand.

Animals↗
PubMed1989

The zygotic control of Drosophila pair-rule gene expression. II. Spatial repression by gap and pair-rule gene products.

We examined gene expression patterns in certain single and double pair-rule mutant embryos to determine which of the largely repressive pair-rule gene interactions are most likely to be direct and which interactions are probably indirect. From these studies we conclude that: (i) hairy+ and even-skipped (eve+) regulate the fushi tarazu (ftz) gene; (ii) eve+ and runt+ regulate the hairy gene; (iii) runt+ regulates the eve gene; but, (iv) runt does not regulate the ftz gene pattern, and hairy does not regulate the eve gene pattern. These pair-rule interactions are not sufficient, however, to explain the periodicity of the hairy and eve patterns, so we examined specific gap gene mutant combinations to uncover their regulatory effects on these two genes. Our surprising observation is that the hairy and eve genes are expressed in embryos where the three key gap genes hunchback (hb), Krüppel (Kr), and knirps (kni) have been removed, indicating that these gap genes are not essential to activate the pair-rule genes. In fact, we show that in the absence of either hb+ or kni+, or both gap genes, the Kr+ product represses hairy expression. These results suggest that gap genes repress hairy expression in the interstripe regions, rather than activate hairy expression in the stripes. The molecular basis of pair-rule gene regulation by gap genes must involve some dual control mechanisms such that combinations of gap genes affect pair-rule transcription in a different manner than a single gap gene.

Animals↗
PubMed1988

Maternal-effect genes that alter the fate map of the Drosophila blastoderm embryo.

The pattern of segmentation in the Drosophila embryo is controlled by at least 25 zygotically active genes and at least 20 maternally active genes. We have examined the pattern of expression of the protein product of the zygotically active segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage in progeny of mutant females homozygous for each of six maternal-effect segmentation genes to observe the early effects of the maternal-effect genes on zygotic gene expression. The genes included exuperantia (a member of the anterior class of maternal-effect segmentation genes); staufen and vasa (members of the posterior class); and torso, trunk, and fs(1)N (members of the terminal class). Mutations in the genes caused a disruption of the normal pattern of ftz stripes in regions of the embryo where gene activity is known to be required. The ftz stripes provide a marker for segmental determination at the cellular blastoderm stage, making it possible to correlate aberrant patterns of ftz protein with defects in cuticle morphology at the end of embryogenesis. ftz protein expression in progeny of females mutant for combinations of the above genes was also examined. The changes in the ftz pattern in progeny of females doubly mutant for genes of the anterior and terminal classes or of the posterior and terminal classes can largely be understood as the result of the additive effects of the single mutations. In contrast, clearly nonadditive effects on the ftz pattern were seen when a mutation in a gene of the anterior class (exuperantia) was combined with mutations in posterior class genes.

Abdomen↗
PubMed1988

Temporal and spatial relationships between segmentation and homeotic gene expression in Drosophila embryos: distributions of the fushi tarazu, engrailed, Sex combs reduced, Antennapedia, and Ultrabithorax proteins.

The specification of segment number and identity in the Drosophila embryo requires the activity of several classes of genes that may be grouped according to the array of pattern elements that they control. Double-label immunofluorescence was used to simultaneously localize the products of genes representative of the pair-rule segmentation class (fushi tarazu), the segment polarity class (engrailed), and the homeotic class (Sex combs reduced, Antennapedia, and Ultrabithorax) of pattern-regulating genes. The temporal order of appearance of each class of proteins and the precise spatial relationships between the products of the different genes are described with single-cell resolution. Boundaries of gene expression, particularly the parasegmental boundaries, are established by early-acting genes such as fushi tarazu and subsequently respected by the expression patterns of later appearing gene products such as engrailed and Ultrabithorax, suggesting regulatory relationships between certain pairs of genes. In addition, the dynamic transitions observed in spatial relationship among the Sex combs reduced, Antennapedia, and Ultrabithorax homeotic protein patterns during the early period of embryogenesis may reflect cross-regulatory interactions among these genes. Finally, some cells contain a single homeotic product, whereas other cells simultaneously contain several, suggesting that certain cells may be determined by the combinatorial action of homeotic genes.

Animals↗
PubMed1988

Expression, function, and regulation of the hairy segmentation protein in the Drosophila embryo.

The Drosophila pair-rule segmentation gene, hairy, is required for the proper development of alternate embryonic segment primordia and the normal spatial expression of another pair-rule gene, fushi tarazu (ftz). We demonstrate that the product of the hairy gene is localized to the nuclei of cells in eight distinct regions of the early embryo. The major stripes of protein accumulation comprise the posterior half and the adjacent anterior portion of alternate segment primordia; the hairy pattern overlaps transiently with ftz expression in a narrow band of cells. Unlike other pair-rule products that have been studied, hairy is not expressed in the embryonic nervous system. The spatial distribution of the nuclear hairy protein and the pattern of ftz expression in embryos lacking hairy function suggests that hairy plays a direct role in repressing ftz gene expression. The pattern of hairy expression in gap gene mutants that affect ftz expression indicates that the gap genes largely act indirectly upon ftz through their effects on hairy.

Animals↗
PubMed1987

The expression and regulation of Sex combs reduced protein in Drosophila embryos.

Homeotic genes are expressed in spatially precise patterns during Drosophila development to control segmental differentiation. The Sex combs reduced (Scr) gene of the Antennapedia gene complex is involved in the determination of the labial and prothoracic segments of the embryo. To study both the wild-type pattern of Scr expression and the regulatory relationships of Scr to other regulatory genes, an antibody probe that detects the Scr protein was prepared. We find that the Scr gene product is expressed in a dynamic pattern over the course of embryogenesis, beginning in the ectoderm in parasegment 2 while the germ band is elongated and extending to parasegment 3 during the completion of germ band shortening. The locations of Scr protein correlate well with the part of the embryo that are altered in Scr- mutants. After head involution occurs, Scr protein is also expressed in the ganglion corresponding to parasegment 2 of the ventral nervous system. The precise spatial expression of Scr is attained through regulation by both homeotic genes and segmentation genes. The lack of proper Antennapedia or Polycomb gene function causes ectopic Scr protein expression. Mutations in the segmentation genes fushi tarazu, hunchback, Krüppel, and giant alter the spatial pattern of Scr expression.

Animals↗
PubMed1987

Genes that control dorsoventral polarity affect gene expression along the anteroposterior axis of the Drosophila embryo.

At least 13 genes control the establishment of dorsoventral polarity in the Drosophila embryo and more than 30 genes control the anteroposterior pattern of body segments. Each group of genes is thought to control pattern formation along one body axis, independently of the other group. We have used the expression of the fushi tarazu (ftz) segmentation gene as a positional marker to investigate the relationship between the dorsoventral and anteroposterior axes. The ftz gene is normally expressed in seven transverse stripes. Changes in the striped pattern in embryos mutant for other genes (or progeny of females homozygous for maternal-effect mutations) can reveal alterations of cell fate resulting from such mutations. We show that in the absence of any of ten maternal-effect dorsoventral polarity gene functions, the characteristic stripes of ftz protein are altered. Normally there is a difference between ftz stripe spacing on the dorsal and ventral sides of the embryo; in dorsalized mutant embryos the ftz stripes appear to be altered so that dorsal-type spacing occurs on all sides of the embryo. These results indicate that cells respond to dorsoventral positional information in establishing early patterns of gene expression along the anteroposterior axis and that there may be more significant interactions between the different axes of positional information than previously determined.

Animals↗
PubMed1986

The localization and regulation of Antennapedia protein expression in Drosophila embryos.

The homeotic Antennapedia (Antp) gene of Drosophila is required for the normal differentiation of the thoracic segments during embryonic development and metamorphosis. Antibodies to a recombinant Antp protein were used to localize the protein in whole mount embryos. Antp is expressed in the nuclei of cells of the thoracic embryonic epidermis and several segments of the ventral and peripheral nervous systems. Analysis of Antp expression in mutant embryos revealed three levels of Antp regulation by genes of the bithorax complex, pleiotropic homeotic loci, and Antp itself. The distributions of the Antp and the Ultrabithorax (Ubx) proteins in doubly-labeled embryos suggest that the Ubx protein may be one direct negative regulator of Antp gene expression.

Animals↗
PubMed1986

Zygotically active genes that affect the spatial expression of the fushi tarazu segmentation gene during early Drosophila embryogenesis.

The establishment of the segmental body pattern of Drosophila requires the coordinated functions of three classes of zygotically active genes early in development. We have examined the effects of mutations in these genes on the spatial expression of the fushi tarazu (ftz) pair-rule segmentation gene. Mutations in four gap loci and in three pair-rule loci dramatically affect the initial pattern of transverse stripes of ftz-containing nuclei. Five other pair-rule genes and several other loci that affect the larval cuticular pattern do not detectably affect ftz expression. No simple regulatory relationships can be deduced. Rather, expression of the ftz gene depends upon the interactions among the different segmentation genes active at each position along the anterior-posterior axis of the early embryo.

Animals↗
PubMed1985

Localization of the fushi tarazu protein during Drosophila embryogenesis.

The fushi tarazu (ftz) gene of Drosophila acts early in embryogenesis to regulate body segmentation. The localization of the ftz protein product in embryos was examined using indirect immunofluorescence microscopy. Antibodies were prepared against a beta-galactosidase-ftz hybrid protein made in E. coli. The ftz protein was first detectable in blastoderm-stage embryos as seven stripes of nuclei encircling the embryos transversely. The stripes persist through the early events of gastrulation, but disappear before overt segmentation is visible. The ftz protein is expressed a second time in some nuclei of the developing nervous system. In contrast to the early pattern, at the later stage, ftz is expressed in each of fifteen metameric subunits of the embryo.

Animals↗
PubMed1984

Paramagnetic 1H and 13C NMR studies on cobalt-substituted human carbonic anhydrase I carboxymethylated at active site histidine-200: molecular basis for the changes in catalytic properties induced by the modification.

Using bromo[1-13C]acetate to modify N tau of His-200 of human carbonic anhydrase isozyme I leads to the introduction of a useful 13C NMR probe into the active site. To complement our previous diamagnetic NMR studies with this probe, we have now succeeded in directly observing the paramagnetically perturbed resonance of the carboxylate in the cobalt-substituted modified enzyme above pH 8. In the pH range 8-10, the resonance undergoes a pH-dependent slow-exchange process, with the more alkaline form having a much smaller pseudocontact shift and a narrower line width. Below pH 8, the resonance apparently undergoes a very large paramagnetic downfield shift that was estimated by extrapolation. An ionization of approximate pK of 6 appears to control this process. Paramagnetic spin-relaxation studies on the resonance under conditions where it was directly observed yielded distance measurements between the carboxylate carbon and the active site cobalt ion. In inhibitor complexes, this distance was in the range of 5-7 A. In the absence of inhibitors, the distance was approximately 3.0-3.2 A at pH 7.9, consistent with the coordination of the carboxylate to the metal. However, at pH 10, the distance was increased to 4.8 A. These distance determinations were aided by relaxation measurements of a paramagnetically shifted proton resonance at 60-65 ppm downfield assigned by others to a proton of a ligand histidine of metal and confirmed by us to be 5.2 +/- 0.1 A from the metal. Our findings provide a molecular basis for the observed changes in catalytic properties that accompany the carboxymethylation.

Acetates↗
PubMed1983

Conservation of a DNA-binding site in the largest subunit of eukaryotic RNA polymerase II.

Using a monoclonal antibody to a DNA-binding site of calf RNA polymerase II, we found that this site occurs on the largest subunit and is structurally similar in RNA polymerase II of widely divergent eukaryotes. In immuno-blotting of electrophoretically separated subunits, the monoclonal antibody recognized a determinant on the largest polypeptide of all RNA eukaryotic polymerase II forms tested, with a preference for the IIA enzyme subunit of 215 X 10(3) Mr over the partially proteolyzed 180 X 10(3) Mr form. This site is conserved on human, chicken, Drosophila, wheat germ and yeast RNA polymerase II, all of which reacted strongly with the monoclonal antibody. These results contrasted with those obtained with polyclonal antibodies to non-functional determinants of the calf enzyme. The reactivity of the polyclonal antibody with eukaryotic RNA polymerase II steadily decreased with increasing evolutionary distance from the original antigen; the yeast enzyme showed no cross-reactivity. These results suggest that a basic functional feature of eukaryotic RNA polymerase II has been strongly conserved and support the view that divergence of RNA polymerase II has taken place mainly in other, perhaps regulatory, sites of the enzyme.

Animals↗
PubMed1983

Antibodies to calf thymus RNA polymerase II from egg yolks of immunized hens.

Polyclonal antibodies to calf thymus RNA polymerase II were raised in laying hens. Up to 75 mg of immunoglobulin/egg yolk were extracted by the polyethylene glycol procedure of Roeder (Roeder, R.G. (1976) in RNA Polymerase (Losick, R., and Chamberlin, M., eds) pp. 285-330, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The concentration of specific antibody in egg yolks (IgY) was comparable to that of serum as measured by enzyme-linked immunoassay. Purified antibody was shown to be directed against enzyme by removal of enzyme activity in immune complexes precipitated by rabbit anti-chicken IgY. The antibodies recognized several of the subunits of the enzyme as determined by their reactivity with polypeptides transferred to nitrocellulose paper after gradient sodium dodecyl sulfate-gel electrophoresis. Production of antibodies in laying hens may facilitate the study of other highly conserved antigens that are poorly immunogenic in mammalian hosts.

Animals↗
PubMed1982

Inhibitory monoclonal antibody to calf thymus RNA polymerase II blocks formation of enzyme-DNA complexes.

Monoclonal antibodies were prepared against purified calf thymus RNA polymerase II (RNA nucleotidyltransferase, EC 2.7.7.6). Two distinct antibodies were identified on the basis of their ability to bind RNA polymerase II in a solid-phase assay and to remove enzyme activity from solution in complexes precipitated by anti-immunoglobulin antibodies. One monoclonal antibody directly inhibited enzyme activity in solution. Detailed study of the mechanism of enzyme inhibition by this antibody revealed that it prevented the formation of enzyme-DNA complexes and had no effect on chain elongation. Inhibition of DNA binding and catalytic activity depended on preincubation of antibody with enzyme and was irreversible. Without preincubation, the enzyme readily bound to DNA in the presence of antibody, indicating that the binding of the free enzyme to DNA is faster than that of the antibody to the enzyme. In a competitive immunoassay enzyme-DNA complexes were much less reactive with antibody than was free enzyme, suggesting that DNA either blocks the antigenic site or alters the enzyme's conformation, preventing antibody recognition. These antibodies may serve as useful reagents for analysis of RNA polymerase structure and function.

Animals↗