Isolation and expression of two novel Wnt/wingless gene homologues in Drosophila

Development ◽  
1992 ◽  
Vol 115 (2) ◽  
pp. 475-485 ◽  
Author(s):  
J. Russell ◽  
A. Gennissen ◽  
R. Nusse
Keyword(s):  
Molecular Mass ◽  
Limb Development ◽  
Expression Patterns ◽  
Relative Molecular Mass ◽  
Gene Products ◽  
Chromosome 2 ◽  
Amino Terminal ◽  
Drosophila Embryogenesis ◽  
Segment Polarity ◽  
Carboxy Terminal

Wingless (wg), the Drosophila homologue of the mouse Wnt-1 proto-oncogene, is a segment polarity gene essential in each segment for normal Drosophila development. We here report the isolation of two novel Drosophila Wnt homologues, DWnt-2 and DWnt-3, and thus the existence of a Wnt/wingless gene family in Drosophila. DWnt-2 and DWnt-3 map to chromosome 2 position 45E and chromosome X position 17A/B, respectively. DWnt-2 and DWnt-3, like the other known Wnt genes, encode amino-terminal signal peptides suggesting that the gene products are secreted proteins. The putative translation product of DWnt-2 and the carboxy-terminal half of the deduced DWnt-3 product are both rich in conserved cysteine residues. In comparison with other Wnt gene products, mostly about 40 × 10(3) relative molecular mass, the DWnt-3 protein has an extended amino terminus and a long internal insert, and its predicted relative molecular mass is 113 × 10(3). The expression patterns of these two Wnt/wg homologues are dynamic during Drosophila embryogenesis. The distribution of DWnt-2 transcripts is predominantly segmented, with the additional presence of transcripts in the presumptive gonads. Transcripts of both DWnt-2 and DWnt-3 appear to be associated with limb primordia in the embryo and may therefore specify limb development. DWnt-3 is also expressed in mesodermal and neurogenic regions. The distribution of DWnt-3 transcripts in cells of the central nervous system (CNS) during Drosophila embryogenesis suggests that DWnt-3 could be involved in CNS development.

Dose-dependent regulation of pair-rule stripes by gap proteins and the initiation of segment polarity

Development ◽  
1990 ◽  
Vol 110 (3) ◽  
pp. 759-767 ◽  
Author(s):  
R. Warrior ◽  
M. Levine
Keyword(s):  
Expression Patterns ◽  
Gene Products ◽  
Embryonic Cells ◽  
Periodic Patterns ◽  
Threshold Response ◽  
Gap Gene ◽  
Relative Placement ◽  
Segment Polarity ◽  
Pair Rule Gene ◽  
Pair Rule

A key step in Drosophila segmentation is the establishment of periodic patterns of pair-rule gene expression in response to gap gene products. From an examination of the distribution of gap and pair-rule proteins in various mutants, we conclude that the on/off periodicity of pair-rule stripes depends on both the exact concentrations and combinations of gap proteins expressed in different embryonic cells. It has been suggested that the distribution of gap gene products depends on cross-regulatory interactions among these genes. Here we provide evidence that autoregulation also plays an important role in this process since there is a reduction in the levels of Kruppel (Kr) RNA and protein in a Kr null mutant. Once initiated by the gap genes each pair-rule stripe is bell shaped and has ill-defined margins. By the end of the fourteenth nuclear division cycle, the stripes of the pair-rule gene even-skipped (eve) sharpen and polarize, a process that is essential for the precisely localized expression of segment polarity genes. This sharpening process appears to depend on a threshold response of the eve promoter to the combinatorial action of eve and a second pair-rule gene hairy. The eve and hairy expression patterns overlap but are out of register and the cells of maximal overlap form the anterior margin of the polarized eve stripe. We propose that the relative placement of the eve and hairy stripes may be an important factor in the initiation of segment polarity.

cDNA cloning and immunolocalization of a Na(+)-H+ exchanger in LLC-PK1 renal epithelial cells

1991 ◽  
Vol 261 (6) ◽  
pp. F1088-F1094 ◽  
Author(s):  
R. F. Reilly ◽  
F. Hildebrandt ◽  
D. Biemesderfer ◽  
C. Sardet ◽  
J. Pouyssegur ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Human Growth ◽  
Relative Molecular Mass ◽  
Hydrophobic Region ◽  
Amino Terminal ◽  
Established Line ◽  
Hydrophilic Region ◽  
Antipeptide Antibody ◽  
Carboxy Terminal ◽  
Hydrophilic Domain

LLC-PK1 cells, an established line from pig kidney, express basolateral and apical Na(+)-H+ exchangers that can be distinguished by their different sensitivities to the amiloride analogue, N-ethyl-N-isopropylamiloride. In the present study, the polymerase chain reaction (PCR) and library screening were used to clone a cDNA for one of the exchangers, based on homology with the recently isolated cDNA for a human growth factor-activatable Na(+)-H+ exchanger. There proved to be significant homology between the LLC-PK1 and human sequences, with nucleotide identities of 75, 93, and 85% in the 5'-untranslated, coding, and 3'-untranslated regions, respectively. The LLC-PK1 cDNA encodes a predicted protein of 818 amino acids with a relative molecular mass of 90,999, consisting of an amino-terminal hydrophobic region and a carboxy-terminal hydrophilic region; its deduced amino acid sequence shows 95% identity with that of the human protein. To investigate the localization of the encoded protein, antisera were generated against a synthetic oligopeptide from the hydrophobic region and a fusion protein from the carboxy-terminal hydrophilic domain. Indirect immunofluorescence and confocal microscopy revealed that the antisera labeled the basolateral but not the apical membrane of confluent LLC-PK1 cells. Labeling by the antipeptide antibody was specifically blocked by preincubation with the synthetic peptide and coincided exactly with the pattern produced by a monoclonal antibody against Na(+)-K(+)-ATPase. Thus, the LLC-PK1 cDNA encodes the basolateral Na(+)-H+ exchanger, which must differ structurally from the apical form, at least in the region of the oligopeptide and the fusion protein.

The Rubisco large subunit binding protein

1986 ◽  
Vol 313 (1162) ◽  
pp. 419-428 ◽  
Keyword(s):  
Pisum Sativum ◽  
Molecular Mass ◽  
Binding Protein ◽  
Large Subunit ◽  
Sodium Dodecyl ◽  
Relative Molecular Mass ◽  
Beta Subunits ◽  
Higher Plant ◽  
Amino Terminal ◽  
Intact Chloroplasts

Newly synthesized Rubisco large subunits made by isolated intact chloroplasts from Pisum sativum are bound non-covalently to another protein, termed the Rubisco large subunit binding protein. This protein is implicated in the assembly of Rubisco in higher plant chloroplasts. The binding protein has been purified from Pisum sativum in the form of an oligomer of relative molecular mass ( M r ) about 720000. Analysis on polyacrylamide gels containing sodium dodecyl sulphate reveals equal amounts of two different types of subunit, termed alpha ( M r about 61000) and beta ( M r about 60000); thus the oligomer has the composition a α 6 β 6 . The alpha and beta subunits have been separated; their amino-terminal sequences are different, and antibodies raised against one subunit do not cross-react with the other subunit. Antibodies raised against the binding protein do not cross-react with the Rubisco large subunit, but do cross-react with polypeptides of M r about 60000 in extracts of chloroplasts from wheat, barley and tobacco, and in extracts of leucoplasts from castor-bean endosperm. The binding protein is made as a higher-molecular-mass precursor whan leaf polysomes are translated in a wheatgerm extract containing chloramphenicol, but is not synthesized by isolated intact chloroplasts. Thus the binding protein subunits are synthesized by cytoplasmic ribosomes and hence are likely to be encoded by nuclear genes. Etiolated Pisum plants contain binding protein, but exposure to light does not cause the same dramatic increase in amount that is seen in the case of Rubisco. Treatment of stromal extracts with Mg-ATP in the range 0.1-5.0 mM causes dissociation of the binding protein oligomer into monomeric subunits; CTP, GTP, UTP, AMP and cyclic AMP do not have this effect. Mg 2+ is required for dissociation but can be replaced by Ca 2+ . Newly synthesized large subunits are released when the binding protein oligomer is dissociated, but re-attach when the dissociation is reversed by removal of ATP.

scholarly journals Identification of the ligand-binding domains of CD22, a member of the immunoglobulin superfamily that uniquely binds a sialic acid-dependent ligand.

1995 ◽  
Vol 181 (4) ◽  
pp. 1581-1586 ◽  
Author(s):  
P Engel ◽  
N Wagner ◽  
A S Miller ◽  
T F Tedder
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Sialic Acid ◽  
Ligand Binding ◽  
Molecular Mass ◽  
Immunoglobulin Superfamily ◽  
Relative Molecular Mass ◽  
Sequence Motif ◽  
Amino Terminal ◽  
Ig Superfamily

CD22 is a B cell-restricted member of the immunoglobulin (Ig) superfamily that functions as an adhesion receptor for leukocytes and erythrocytes. CD22 is unique among members of the Ig superfamily in that it has been suggested to bind a series of sialic acid-dependent ligands, potentially through different functional domains expressed by different splice variants of CD22. In this study, the epitopes identified by a large panel of function-blocking and non-function-blocking CD22 monoclonal antibodies were localized to specific Ig-like domains, revealing that all function-blocking monoclonal antibodies bound to the first and/or second Ig-like domains. Consistent with a single ligand-binding region, the two amino-terminal domains were the functional unit that mediated CD22 adhesion with lymphocytes, neutrophils, monocytes, and erythrocytes. The predominant cell surface species of CD22 was a full length 140,000 relative molecular mass seven Ig-like domain glycoprotein and a minor 130,000 relative molecular mass form lacking the fourth domain. While the two amino-terminal Ig-like domains of CD22 are structurally similar to those found in other members of the Ig superfamily involved in cell adhesion and containing an amino acid sequence motif associated with integrin recognition, site-directed mutagenesis of critical residues surrounding this motif did not disrupt CD22-mediated adhesion. These results demonstrate that the unique ligand-binding properties of CD22 are distinct from those of other members of the Ig superfamily involved in integrin-mediated cell adhesion.

scholarly journals Structure and Function Analysis of LIN-14, a Temporal Regulator of Postembryonic Developmental Events in Caenorhabditis elegans

2000 ◽  
Vol 20 (6) ◽  
pp. 2285-2295 ◽  
Author(s):  
Yang Hong ◽  
Rosalind C. Lee ◽  
Victor Ambros
Keyword(s):  
Caenorhabditis Elegans ◽  
Function Analysis ◽  
Cell Types ◽  
Terminal Region ◽  
Gene Products ◽  
Amino Terminal ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Terminal Domains

ABSTRACT During postembryonic development of Caenorhabditis elegans, the heterochronic gene lin-14 controls the timing of developmental events in diverse cell types. Three alternativelin-14 transcripts are predicted to encode isoforms of a novel nuclear protein that differ in their amino-terminal domains. In this paper, we report that the alternative amino-terminal domains of LIN-14 are dispensable and that a carboxy-terminal region within exons 9 to 13 is necessary and sufficient for in vivo LIN-14 function. A transgene capable of expressing only one of the three alternativelin-14 gene products rescues a lin-14 null mutation and is developmentally regulated by lin-4. This shows that the deployment of alternative lin-14 gene products is not critical for the ability of LIN-14 to regulate downstream genes in diverse cell types or for the in vivo regulation of LIN-14 level by lin-4. The carboxy-terminal region of LIN-14 contains an unusual expanded nuclear localization domain which is essential for LIN-14 function. These results support the view that LIN-14 controls developmental timing in C. elegans by regulating gene expression in the nucleus.

scholarly journals Ulnaless (Ul), a regulatory mutation inducing both loss-of-function and gain-of-function of posterior Hoxd genes

Development ◽  
1997 ◽  
Vol 124 (18) ◽  
pp. 3493-3500 ◽  
Author(s):  
Y. Herault ◽  
N. Fraudeau ◽  
J. Zakany ◽  
D. Duboule
Keyword(s):  
Limb Development ◽  
Control Mechanism ◽  
Expression Patterns ◽  
Appendicular Skeleton ◽  
Chromosome 2 ◽  
Loss Of Function ◽  
Regulatory Mutation ◽  
Dominant Mutation ◽  
X Ray ◽  
Limb Morphogenesis

Ulnaless (Ul), an X-ray-induced dominant mutation in mice, severely disrupts development of forearms and forelegs. The mutation maps on chromosome 2, tightly linked to the HoxD complex, a cluster of regulatory genes required for proper morphogenesis. In particular, 5′-located (posterior) Hoxd genes are involved in limb development and combined mutations within these genes result in severe alterations in appendicular skeleton. We have used several engineered alleles of the HoxD complex to genetically assess the potential linkage between these two loci. We present evidence indicating that Ulnaless is allelic to Hoxd genes. Important modifications in the expression patterns of the posterior Hoxd-12 and Hoxd-13 genes at the Ul locus suggest that Ul is a regulatory mutation that interferes with a control mechanism shared by multiple genes to coordinate Hoxd function during limb morphogenesis.

scholarly journals An amino-terminal extension is required for the secretion of chick agrin and its binding to extracellular matrix.

1995 ◽  
Vol 131 (6) ◽  
pp. 1547-1560 ◽  
Author(s):  
A J Denzer ◽  
M Gesemann ◽  
B Schumacher ◽  
M A Ruegg
Keyword(s):  
Amino Acids ◽  
Extracellular Matrix ◽  
Amino Acid ◽  
Molecular Mass ◽  
Motor Neurons ◽  
Acetylcholine Receptors ◽  
Relative Molecular Mass ◽  
Amino Terminal ◽  
Tissue Homogenates ◽  
Receptor Clusters

Agrin is an extracellular matrix (ECM) protein with a calculated relative molecular mass of more than 200 kD that induces the aggregation of acetylcholine receptors (AChRs) at the neuromuscular junction. This activity has been mapped to its COOH terminus. In an attempt to identify the functions of the NH2-terminal end, we have now characterized full-length chick agrin. We show that chick agrin encoded by a previously described cDNA is not secreted from transfected cells. Secretion is achieved with a construct that includes an additional 350 bp derived from the 5' end of chick agrin mRNA. Recombinant agrin is a heparan sulfate proteoglycan (HSPG) of more than 400 kD with glycosaminoglycan side chains attached only to the NH2-terminal half. Endogenous agrin in tissue homogenates also has an apparent molecular mass of > 400 kD. While the amino acid sequence encoded by the 350-bp extension has no homology to published rat agrin, it includes a stretch of 15 amino acids that is 80% identical to a previously identified bovine HSPG. The extension is required for binding of agrin to ECM. AChR aggregates induced by recombinant agrin that includes the extension are considerably smaller than those induced by agrin fragments, suggesting that binding of agrin to ECM modulates the size of receptor clusters. In addition, we found a site encoding seven amino acids at the NH2-terminal end of agrin that is alternatively spliced. While motor neurons express the splice variant with the seven amino acid long insert, muscle cells mainly synthesize isoforms that lack this insert. In conclusion, the cDNAs described here code for chick agrin that has all the characteristics previously allocated to endogenous agrin.

scholarly journals Alpha-adaptin, a marker for endocytosis, is expressed in complex patterns during Drosophila development.

1997 ◽  
Vol 8 (8) ◽  
pp. 1391-1403 ◽  
Author(s):  
S Dornan ◽  
A P Jackson ◽  
N J Gay
Keyword(s):  
Nervous System ◽  
Neuromuscular Junctions ◽  
Single Gene ◽  
Expression Patterns ◽  
Homeobox Gene ◽  
Coated Vesicle ◽  
Chromosome 2 ◽  
Amino Terminal ◽  
Visceral Mesoderm ◽  
Wide Range

A Drosophila cDNA encoding a structural homologue of the mammalian coated vesicle component alpha-adaptin (AP2 adaptor complex) has been cloned and sequenced. The mammalian and invertebrate sequences are highly conserved, especially within the amino terminal region, a domain that mediates interactions with other components within the AP2 complex and with specific receptors tails. Mammalian alpha-adaptins are encoded by two genes; however, Drosophila alpha-adaptin has a single gene locus, within polytene bands 21C2-C3 on the left arm of the chromosome 2, closely adjacent to the paired homeobox gene aristaless. There seem to be at least two Drosophila alpha-adaptin transcripts expressed, plausibly by alternative splicing. One of the transcripts is more abundant during early embryogenesis and may be of maternal origin. We have studied the distribution of the alpha-adaptin protein throughout embryogenesis and at the neuromuscular junction of the third instar larva. During cellularization of the blastoderm embryo, the protein is seen between and ahead of the elongating nuclei, and then redistributes to the cell surface during gastrulation. These observations suggest a role for endocytosis in cellularization and are consistent with the finding that dynamin (the shibire gene product), another component of the endocytic mechanism, is required for cellularization. At later stages of embryogenesis, alpha-adaptin is expressed in complex and dynamic patterns. It is strongly induced in elements of the central and peripheral nervous system (e.g., in neuroblasts, the presumptive stomatogastric nervous system, and the lateral chordotonal sense organs), in the Garland cells, the adult midgut precursors, the antenno-maxillary complex, the endoderm, the fat bodies, and the visceral mesoderm. In the larva, alpha-adaptin is localized at the plasma membrane in the synaptic boutons of the neuromuscular junctions. The cells expressing high levels of alpha-adaptin are known or expected to support high levels of endocytosis; thus, this coated vesicle protein seems to be an excellent marker for endocytic activity. The expression patterns of dynamin, detected in the embryo by in situ hybridization methods, are very similar to those reported here for alpha-adaptin reflecting the likely coordinated expression of endocytic components. Taken together with previous evidence, our results suggest that endosomal vesicle trafficking, membrane recycling, and the regulation of endocytosis play critical roles in the wide range of developmental processes.

Analysis of the gooseberry locus in Drosophila embryos: gooseberry determines the cuticular pattern and activates gooseberry neuro

Development ◽  
1993 ◽  
Vol 118 (1) ◽  
pp. 21-31 ◽  
Author(s):  
T. Gutjahr ◽  
N.H. Patel ◽  
X. Li ◽  
C.S. Goodman ◽  
M. Noll
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Expression Patterns ◽  
P Element ◽  
Gene Products ◽  
Cell Fates ◽  
Segment Polarity ◽  
The Central Nervous System ◽  
Characteristic Segment ◽  
Drosophila Embryos

The segment-polarity class of segmentation genes in Drosophila are primarily involved in the specification of sub-segmental units. In addition, some of the segment-polarity genes have been shown to specify cell fates within the central nervous system. One of these loci, gooseberry, consists of two divergently transcribed genes, gooseberry and gooseberry neuro, which share a paired box as well as a paired-type homebox. Here, the expression patterns of the two gooseberry gene products are described in detail. The gooseberry protein appears in a characteristic segment-polarity pattern of stripes at gastrulation and persists until head involution. It is initially restricted to the ectodermal and neuroectodermal germ layer, but is later detected in mesodermal and neuronal cells as well. The gooseberry neuro protein first appears during germ band extension in cells of the central nervous system and also, much later, in epidermal stripes and in a small number of muscle cells. P-element-mediated transformation with the gooseberry gene has been used to demonstrate that gooseberry transactivates gooseberry neuro and is sufficient to rescue the gooseberry cuticular phenotype in the absence of gooseberry neuro.

Extraction of polyethylene glycols with dicarbolide solutions in nitrobenzene

1990 ◽  
Vol 55 (8) ◽  
pp. 1959-1967 ◽  
Author(s):  
Petr Vaňura ◽  
Pavel Selucký
Keyword(s):  
Polyethylene Glycol ◽  
Molecular Mass ◽  
Extraction Constant ◽  
Metal Extraction ◽  
Polyethylene Glycols ◽  
Relative Molecular Mass ◽  
Published Data ◽  
Average Molecular Mass ◽  
Peg 400 ◽  
Extraction Constants

The extraction of polyethylene glycol of average molecular mass 400 (PEG 400) with dicarbolide solution in nitrobenzene and of longer-chain polyethylene glycol, of average molecular mass 1 500 (PEG 1 500), with chlorinated dicarbolide solution in nitrobenzene was studied. During the extraction of PEG 400, the polyethylene glycol solvates the Horg+ ion in the organic phase giving rise to the HLorg+ species (L is polyethylene glycol). The obtained value of the extraction constant Kex(HLorg+) = 933 is consistent with published data of metal extraction. Extraction of PEG 1 500 was treated applying the simplified assumption that the thermodynamic behaviour of PEG 1 500 is the same as that of n molecules of polyethylene glycol with relative molecular mass 1 500/n, each solvating one cation. For this model, the value of n = 3.2 ± 1.1 and the values of the extraction constants of the HL1/n,org+ and HL2/n,org+ species were obtained by using the adapted program LETAGROP. This value of n is consistent with published extraction data in the presence of polyethylene glycol with a relative molecular mass from 200 to 1 000.

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