Two distinct mechanisms for differential positioning of gene expression borders involving the Drosophila gap protein giant

We have combined genetic experiments and a targeted misexpression approach to examine the role of the gap gene giant (gt) in patterning anterior regions of the Drosophila embryo. Our results suggest that gt functions in the repression of three target genes, the gap genes Krüppel (Kr) and hunchback (hb), and the pair-rule gene even-skipped (eve). The anterior border of Kr, which lies 4-5 nucleus diameters posterior to nuclei that express gt mRNA, is set by a threshold repression mechanism involving very low levels of gt protein. In contrast, gt activity is required, but not sufficient for formation of the anterior border of eve stripe 2, which lies adjacent to nuclei that express gt mRNA. We propose that gt's role in forming this border is to potentiate repressive interaction(s) mediated by other factor(s) that are also localized to anterior regions of the early embryo. Finally, gt is required for repression of zygotic hb expression in more anterior regions of the embryo. The differential responses of these target genes to gt repression are critical for the correct positioning and maintenance of segmentation stripes, and normal anterior development.     

Comparison of the structure and expression of odd-skipped and two related genes that encode a new family of zinc finger proteins in Drosophila

The odd-skipped (odd) gene, which was identified on the basis of a pair-rule segmentation phenotype in mutant embryos, is initially expressed in the Drosophila embryo in seven pair-rule stripes, but later exhibits a segment polarity-like pattern for which no phenotypic correlate is apparent. We have molecularly characterized two embryonically expressed odd-cognate genes, sob and bowel (bowl), that encode proteins with highly conserved C2H2 zinc fingers. While the Sob and Bowl proteins each contain five tandem fingers, the Odd protein lacks a fifth (C-terminal) finger and is also less conserved among the four common fingers. Reminiscent of many segmentation gene paralogues, the closely linked odd and sob genes are expressed during embryogenesis in similar striped patterns; in contrast, the less-tightly linked bowl gene is expressed in a distinctly different pattern at the termini of the early embryo. Although our results indicate that odd and sob are more likely than bowl to share overlapping developmental roles, some functional divergence between the Odd and Sob proteins is suggested by the absence of homology outside the zinc fingers, and also by amino acid substitutions in the Odd zinc fingers at positions that appear to be constrained in Sob and Bowl     

Pair-rule and gap gene mutants in the flour beetle Tribolium castaneum

Early pattern formation in the Drosophila embryo occurs in a syncytial blastoderm where communication between nuclei is unimpeded by cell walls. During the development of other insects, similar gene expression patterns are generated in a cellular environment. In Tribolium, for instance, pair-rule stripes are transiently expressed near the posterior end of the growing germ band. To elucidate how pattern formation in such a situation deviates from that of Drosophila, functional data about the genes involved are essential. In a genetic screen for Tribolium mutants affecting the larval cuticle pattern, we isolated 4 mutants (from a total of 30) which disrupt segmentation in the thorax and abdomen. Two of these mutants display clear pair-rule phenotypes. This demonstrates that not only the expression, but also the function of pair-rule genes in this short-germ insect is in principle similar to Drosophila. The other two mutants appear to identify gap genes. They provide the first evidence for the involvement of gap genes in abdominal segmentation of short-germ embryos. However, significant differences between the phenotypes of these mutants and those of known Drosophila gap mutants exist which indicates that evolutionary changes occurred in either the regulation or action of these genes.     

Functional analysis of eve stripe 2 enhancer evolution in Drosophila: rules governing conservation and change

Experimental investigations of eukaryotic enhancers suggest that multiple binding sites and trans-acting regulatory factors are often required for wild-type enhancer function. Genetic analysis of the stripe 2 enhancer of even-skipped (eve), an important developmental gene in Drosophila, provides support for this view. Given the importance of even-skipped expression in early Drosophila development, it might be predicted that many structural features of the stripe 2 enhancer will be evolutionarily conserved, including the DNA sequences of protein binding sites and the spacing between them. To test this hypothesis, we compared sequences of the stripe 2 enhancer between four species of Drosophila: D. melanogaster, D. yakuba, D. erecta and D. pseudoobscura. Our analysis revealed a large number of nucleotide substitutions in regulatory protein binding sites for bicoid, hunchback, Kruppel and giant, as well as a systematic change in the size of the enhancer. Some of the binding sites in D. melanogaster are either absent or modified in other species. One functionally important bicoid-binding site in D. melanogaster appears to be recently evolved. We, therefore, investigated possible functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation. This analysis revealed that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D. melanogaster embryos. Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native eve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein. We argue that stripe 2 eve expression in Drosophila evolution can be viewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe. We further hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly deleterious and adaptive mutations in regulatory protein binding sites as well as in the spacing between binding sites. This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer.     

Integration of the head and trunk segmentation systems controls cephalic furrow formation in Drosophila

Genetic and molecular analyses of patterning of the Drosophila embryo have shown that the process of segmentation of the head is fundamentally different from the process of segmentation of the trunk. The cephalic furrow (CF), one of the first morphological manifestations of the patterning process, forms at the juxtaposition of these two patterning systems. We report here that the initial step in CF formation is a change in shape and apical positioning of a single row of cells. The anteroposterior position of these initiator cells may be defined by the overlapping expression of the head gap gene buttonhead (btd) and the primary pair-rule gene even-skipped (eve). Re-examination of the btd and eve phenotypes in live embryos indicated that both genes are required for CF formation. Further, Eve expression in initiator cells was found to be dependent upon btd activity. The control of eve expression by btd in these cells is the first indication of a new level of integrated regulation that interfaces the head and trunk segmentation systems. In conjunction with previous data on the btd and eve embryonic phenotypes, our results suggest that interaction between these two genes both controls initiation of a specific morphogenetic movement that separates two morphogenetic fields and contributes to patterning the hinge region that demarcates the procephalon from the segmented germ band.     

Co-amplification of a novel gene, NAG, with the N-myc gene in neuroblastoma

Substantial evidence implicates amplification of the N-myc gene with aggressive tumor growth and poor outcome in neuroblastoma. However some evidence suggests that this gene alone is not the sole determinant of outcome in N-myc amplified tumors. We have searched for genes that co-amplify with N-myc in neuroblastoma by means of two-dimensional analysis of genomic restriction digests. Using this approach, we have identified and cloned a novel genomic fragment which is co-amplified with N-myc in neuroblastomas. This fragment was mapped in close vicinity to N-myc on chromosome arm 2p24. It was amplified in 5/8 N-myc amplified neuroblastoma cell lines and in 9/13 N-myc amplified tumors. Using a PCR-based approach we isolated a 4.5 kb c-DNA sequence that is partly contained in the genomic fragment. The open reading frame of the cDNA encodes a predicted protein of 1353 amino acids (aa). The homology of the predicted protein, which we designated NAG (neuroblastoma amplified gene), to a C. elegans protein of as yet unknown function, and its ubiquitous expression suggest that NAG may serve an essential function. By Northern blot analysis we showed that amplification of the cloned gene correlates with over-expression in neuroblastoma cell lines. Amplification and consequent over-expression of NAG may, therefore, contribute to the phenotype of a subset of neuroblastomas.     

Expression of two even-skipped genes eve1 and evx2 during zebrafish fin morphogenesis and their regulation by retinoic acid

Growth and patterning during fin regeneration depend, like for fin development, on the integrated expression of homeogenes. In the present work we have studied, by in situ hybridization, the expression and regulation of two vertebrate homologs eve1 and evx2 of the Drosophila pair-rule even-skipped gene family. Upon amputation of pectoral and caudal fins, both genes, expressed transiently in the mesenchyme during early stages of fin development of these fins, are turned on. During the formation of the blastema they are transcribed first in the mesenchyme located underneath the wound epidermis and then, their expression is restricted to the regenerating rays regions. These expression patterns are developmentally regulated since both genes are no longer transcribed when the bony rays are differentiating. Exposure of the regenerates to retinoic acid (RA) modifies the boundaries of eve1 and evx2 expression: the signal is down-regulated in the ray region and up-regulated in the interray region. Moreover, expression is induced in the wound epidermis. These results indicate that eve1 and evx2 products are part of the molecular signals involved in pattern formation of the fin and fin rays in connection with outgrowth. RA might alter growth and morphogenesis of the regenerating fins by a fine regulation of these genes among others.     

Molecular characterization of a new type of receptor-like kinase (wlrk) gene family in wheat

In plants, several types of receptor-like kinases (RLK) have been isolated and characterized based on the sequence of their extracellular domains. Some of these RLKs have been demonstrated to be involved in plant development or in the reaction to environmental signals. Here, we describe a RLK gene family in wheat (wlrk, wheat leaf rust kinase) with a new type of extracellular domain. A member of this new gene family has previously been shown to cosegregate with the leaf rust resistance gene Lr10. The diversity of the wlrk gene family was studied by cloning the extracellular domain of different members of the family. Sequence comparisons demonstrated that the extracellular domain consists of three very conserved regions interrupted by three variable regions. Linkage analysis indicated that the wlrk genes are specifically located on chromosome group 1 in wheat and on the corresponding chromosomes of other members of the Triticeae family. The wlrk genes are constitutively expressed in the aerial parts of the plant whereas no expression was detected in roots. Protein immunoblots demonstrated that the WLRK protein coded by the Lrk10 gene is an intrinsic plasma membrane protein. This is consistent with the hypothesis that WLRK proteins are receptor protein kinases localized to the cell surface. In addition, we present preliminary evidence that other disease resistance loci in wheat contain genes which are related to wlrk.     

Missense mutations in the PAX6 gene in aniridia

PURPOSE: Aniridia is caused by a mutation of the PAX6 gene. Haploinsufficiency of the gene product is thought to result in the aniridia phenotype, because most mutations thus far detected have been large deletions encompassing the entire gene and nonsense, frameshift, or splice errors that result in premature translational termination on one of the alleles. Only two missense mutations have been detected in aniridia pedigrees, each of which occurs in its paired domain or homeodomain. In this study, four novel missense mutations were found in three aniridia pedigrees. METHODS: Polymerase chain reaction-single-strand conformation polymorphism analysis and sequencing of the PAX6 gene were performed using genomic DNA of three aniridia pedigrees and more than 100 healthy control subjects. RESULTS: Three mutations occurred in the N-terminal subdomain of the paired domain, namely N17S, I29V, and R44Q, the first two of which were detected on the same allele of one patient. The other mutation (Q178H) was in the linking portion of the paired domain and homeodomain. CONCLUSIONS: These missense mutations give rise to haploinsufficiency by another route, because the missense mutations presented here resulted in an aniridia phenotype indistinguishable from that caused by a heterozygous deletion of the entire PAX6 gene.     

Functionality and cell anchorage dependence of the African swine fever virus gene A179L, a viral bcl-2 homolog, in insect cells

The African swine fever virus gene A179L has been shown to be a functional member of the ced9/bcl-2 family of apoptosis inhibitors in mammalian cell lines. In this work we have expressed the A179L gene product (p21) under the control of the baculovirus polyhedrin promoter using a baculovirus system. Expression of the A179L gene neither altered the baculovirus replication phenotype nor delayed the shutoff of cellular protein synthesis, but it extended the survival of the infected insect cells to very late times postinfection. The increase in cell survival rates correlated with a marked apoptosis reduction after baculovirus infection. Interestingly, prevention of apoptosis was observed when recombinant baculovirus infections were carried out in monolayer cell cultures but not when cells were infected in suspension, suggesting a cell anchorage dependence for p21 function in insect cells. Cell survival was enhanced under optimal conditions of cell attachment and cell-to-cell contact as provided by extracellular matrix components or poly-D-lysine. Since it was observed that cytoskeleton organization varied depending on culture conditions of insect cells (grown in monolayer versus grown in suspension), these results suggested that A179L might regulate apoptosis in insect cells only when the cytoskeletal support of intracellular signaling is maintained upon cell adhesion. Thus, cell shape and cytoskeleton status might allow variations in intracellular transduction of signals related to cell survival in virus-infected cells.     

Scrambler and yotari disrupt the disabled gene and produce a reeler-like phenotype in mice

Formation of the mammalian brain requires choreographed migration of neurons to generate highly ordered laminar structures such as those in the cortices of the forebrain and the cerebellum. These processes are severely disrupted by mutations in reelin which cause widespread misplacement of neurons and associated ataxia in reeler mice. Reelin is a large extracellular protein secreted by pioneer neurons that coordinates cell positioning during neurodevelopment. Two new autosomal recessive mouse mutations, scramble and yotari have been described that exhibit a phenotype identical to reeler. Here we report that scrambler and yotari arise from mutations in mdab1, a mouse gene related to the Drosophila gene disabled (dab). Both scrambler and yotari mice express mutated forms of mdab1 messenger RNA and little or no mDab1 protein. mDab1 is a phosphoprotein that appears to function as an intracellular adaptor in protein kinase pathways. Expression analysis indicates that mdab1 is expressed in neuronal populations exposed to Reelin. The similar phenotypes of reeler, scrambler, yotari and mdab1 null mice indicate that Reelin and mDab1 function as signalling molecules that regulate cell positioning in the developing brain.     

Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila

The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein (Suhw) that binds to gypsy retrovirus insertions between enhancers and promoters. Suhw bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. This implicates Chip in enhancer-promoter communication. We cloned Chip and find that it encodes a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins that bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is present in all nuclei examined and at numerous sites along the salivary gland polytene chromosomes. Embryos without Chip activity lack segments and show abnormal gap and pair-rule gene expression, although no LIM domain proteins are known to regulate segmentation. We conclude that Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. We suggest that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer-promoter interactions.     

hyp gene products in Alcaligenes eutrophus are part of a hydrogenase-maturation system

In Alcaligenes eutrophus H16 the hyp gene complex consists of six open reading frames hypA1, B1, F1, C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NAD-reducing cytoplasmic enzyme (SH) and a membrane-bound electron-transport-coupled protein (MBH). hypB1 and hypF1 were originally considered to form a single open reading frame designated hypB [Dernedde, J., Eitinger, M. & Friedrich, B. (1993) Arch. Microbiol. 159, 545-553]. Re-examination of the relevant sequence identified hypB1 and hypF1 as two distinct genes. Non-polar in-frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild-type strain. The resulting mutants fall into two classes. Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, B1 and F1 deletion mutants (class II) were not impaired in hydrogen metabolism. Class I mutants were unable to grow on hydrogen under autotrophic conditions. The enzymatic activities of SH and MBH were disrupted in all three class I mutants. Immunoblot analysis showed the presence of the H2-activating SH subunit (HoxH) at levels comparable to those observed in the wild-type strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation. Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane. In the wild-type strain, HoxH and the MBH large subunit (HoxG) undergo C-terminal proteolytic processing before attaining enzymatic activity. In class I mutants this maturation was blocked. 63Ni-incorporation experiments identified both hydrogenases as nickel-free apoproteins in these mutants. Although class II mutants bearing deletions in hypA1, B1 and F1 showed no alteration of the wild-type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A. eutrophus.     

The gene for 16S rRNA methyltransferase (ksgA) functions as a multicopy suppressor for a cold-sensitive mutant of era, an essential RAS-like GTP-binding protein in Escherichia coli

Era, a Ras-like GTP-binding protein in Escherichia coli, has been shown to be essential for growth. However, its cellular functions still remain elusive. In this study, a genetic screening of an E. coli genomic library was performed to identify those genes which can restore the growth ability of a cold-sensitive mutant, Era(Cs) (E200K), at a restrictive temperature when expressed in a multicopy plasmid. Among eight suppressors isolated, six were located at 1 min of the E. coli genomic map, and the gene responsible for the suppression of Era(Cs) (E200K) was identified as the ksgA gene for 16S rRNA transmethylase, whose mutation causes a phenotype of resistance to kasugamycin, a translation initiation inhibitor. This is the first demonstration of suppression of impaired function of Era by overproduction of a functional enzyme. A possible mechanism of the suppression of the Era cold-sensitive phenotype by KsgA overproduction is discussed.     

SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development

G protein-coupled receptors trigger the reorganization of the actin cytoskeleton in many cell types, but the steps in this signal transduction cascade are poorly understood. During Dictyostelium development, extracellular cAMP functions as a chemoattractant and morphogenetic signal that is transduced via a family of G protein-coupled receptors, the cARs. In a strain where the cAR2 receptor gene is disrupted by homologous recombination, the developmental program arrests before tip formation. In a genetic screen for suppressors of this phenotype, a gene encoding a protein related to the Wiskott-Aldrich Syndrome protein was discovered. Loss of this protein, which we call SCAR (suppressor of cAR), restores tip formation and most later development to cAR2(-) strains, and causes a multiple-tip phenotype in a cAR2(+) strain as well as leading to the production of extremely small cells in suspension culture. SCAR-cells have reduced levels of F-actin staining during vegetative growth, and abnormal cell morphology and actin distribution during chemotaxis. Uncharacterized homologues of SCAR have also been identified in humans, mouse, Caenorhabditis elegans, and Drosophila. These data suggest that SCAR may be a conserved negative regulator of G protein-coupled signaling, and that it plays an important role in regulating the actin cytoskeleton.