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PubMed1999

Expression of transcription factor c-Rel and apoptosis occurrence in polydactylous and syndactylous limb buds of the talpid3 mutant chick embryo.

The chicken proto-oncogene c-rel encodes a transcription factor of the Rel/NF-kappa B family. We have previously shown that c-rel mRNAs accumulate in different types of apoptotic cells of the chick embryo, especially in mesenchymal cells within the four cell death areas of the limb bud: the anterior and posterior necrotic zones, the opaque patch and the interdigital necrotic zones. This study aimed to further establish the involvement of c-Rel in apoptosis of the developing limb by investigating its expression in the talpid3 mutant which was originally shown to be defective in apoptosis. However, our preliminary examinations highlighted the apparent presence of apoptotic cells in talpid3 embryos. Hence, we performed a systematic study of the occurrence of apoptosis in mutant and control embryos by the TUNEL method. The results revealed that apoptosis does occur in talpid3 embryos but with altered spatial and temporal patterns. This suggests that the talpid3 mutation does not affect a gene involved in apoptosis per se but rather in the determination of the pattern of apoptosis. Neither the expression of c-Rel nor that of its I kappa B alpha inhibitor are grossly modified in talpid3 limb buds, suggesting that the talpid3 mutation does not affect any of these genes. They are mostly expressed in epidermal, endodermal and striated muscle cells in control and in talpid3 limb buds as well. C-Rel was also detected in some scarce mesenchymal cells that could be apoptotic, in both control and mutant embryos. The only slight difference between control and talpid3 limbs lies in the perichondrium which is not fully differentiated in talpid3 embryos: c-Rel and I kappa B alpha are only faintly expressed in talpid3 perichondrial cells, whereas they are both detected in control perichondrial cells. Taken together, these results suggest that c-Rel could participate in several developmental processes, especially in the differentiation of perichondrial cells, besides its already documented involvement in apoptosis and haematopoeisis.

Journal Article↗
PubMed2004

Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development.

In the developing limb, Bmp4 is expressed in the apical ectodermal ridge (AER) and underlying mesoderm. Insight into the function of Bmp4 in limb development has been hampered by the early embryonic lethality of Bmp4 null embryos. We directly investigated Bmp4 using a conditional null allele of Bmp4 and the Prx1(cre) transgene to inactivate Bmp4 in limb bud mesoderm. The limb bud mesoderm of Prx1(cre);Bmp4 mutants was defective in production of Bmp4 but still competent to respond to Bmp signaling. Prx1(cre);Bmp4 mutant embryos had defective digit patterning including hindlimb preaxial polydactyly with posterior digit transformations. The Prx1(cre);Bmp4 mutants also had postaxial polydactyly with digit five duplications. Bmp4 mutant limbs had delayed induction and maturation of the AER that resulted in expanded Shh signaling. Moreover, the AER persisted longer in the Bmp4 mutant limb buds exposing the forming digits to prolonged Fgf8 signaling. Our data show that Bmp4 in limb mesoderm regulates AER induction and maturation and implicate signaling from the AER in regulation of digit number and identity.

Animals↗
PubMed2003

Asymmetric limb malformations in a new transgene insertional mutant, footless.

Six to eight copies of a transgene integrated into mouse chromosome 15 resulting in a new transgene insertional mutant, Footless, presenting with malformations of the limbs, kidney, and soft palate. Homozygotes possess a unique asymmetric pattern of limb truncations. Posterior structures from the autopod and zeugopod of the hindlimbs are missing with left usually more severely affected than right. In contrast, anterior structures are missing from the right forelimbs. The left forelimb is usually normal except for the absence of the distal telephalanges and nails. These structures are absent on all formed digits. In situ hybridization assays examined the expression of Shh, dHand, Msx2, Fgf8, En1, and Lmx1b in mutant limb buds and indicated normal establishment of the anterior/posterior and dorsal/ventral axes of the developing limbs. However, dysmorphology of the apical ectodermal ridge was observed in the mutant limb buds.

Animals↗
PubMed2003

The upstream regulator, Rsr1p, and downstream effectors, Gic1p and Gic2p, of the Cdc42p small GTPase coordinately regulate initiation of budding in Saccharomyces cerevisiae.

BACKGROUND: Cdc42p, a Rho family small GTPase, is essential for budding initiation in the yeast Saccharomyces cerevisiae. The homologous proteins Gic1p and Gic2p (Gic1/2p) are effectors of Cdc42p, but their precise functions remain unknown. Rsr1p/Bud1p is a Ras family small GTPase that controls the selection of the budding site. Previous observations suggested that Rsr1p-GTP recruits Cdc24p, a GDP/GTP exchange factor for Cdc42p, at the incipient bud site. However, this model only addresses how Rsr1p determines the budding site, because the rsr1 mutant normally initiates budding. RESULTS: Here we show that a rsr1 gic1 gic2 mutant fails to initiate budding, resulting in unbudded, large, and multinucleated cells. Expression of a dominant active or dominant negative mutant of RSR1 also inhibited the growth of the gic1 gic2 mutant, suggesting that cycling of Rsr1p between the GTP- and GDP-bound forms is required for budding initiation in the gic1 gic2 mutant. Among the mutations in effectors of CDC42, only the gic1 gic2 mutation demonstrated a synthetic lethal interaction with rsr1. Increased gene dosage of CDC42 suppressed defects in budding initiation of rsr1 gic1 gic2 mutants containing additional mutations in other effectors of CDC42, including BNI1, CLA4 or STE20. The polarized localization of Bni1p-GFP (green fluorescent protein) and Cla4p-GFP was lost after depletion of Gic1p in the rsr1 gic2 mutant. CONCLUSION: We propose that Gic1/2p may stabilize or maintain a complex consisting of Cdc42p-GTP and its effectors at the budding site, which are assembled by the action of the Rsr1p-Cdc24p system.

Adaptor Proteins, Signal Transducing↗
PubMed1991

Amino acid substitutions within the matrix protein of type D retroviruses affect assembly, transport and membrane association of a capsid.

The functional roles of the matrix (MA) protein in the assembly and maturation of retroviruses was investigated with a series of MA mutants of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive type D retrovirus. The mutants we describe here were generated by the introduction of random point mutations within the MA coding domain by use of sodium bisulphite mutagenesis. Studies of these mutants show that the MA protein plays a critical role in three different, sequential events in the final stages of type D retrovirus replication: (i) folding of the gag gene-encoded precursor poly-proteins into a stable conformation for capsid assembly in the cytoplasm of infected cells; (ii) capsid transport from the site of assembly to the plasma membrane; and (iii) capsid association with, and extrusion of the membrane during virus budding. The mutants described here interfere with or block M-PMV replication at each of these stages. Large numbers of preassembled capsids accumulate within the cytoplasm of transport-defective mutant-infected cells, suggesting that transport of M-PMV capsids to the plasma membrane is an active and specific intracellular targeting process. The initial association of the capsid with the membrane may depend upon this intracytoplasmic transport process but additional protein-lipid interactions that involve the MA protein are required for membrane extrusion around the preformed capsids; in cells infected with the budding-defective mutant, assembled capsids accumulate under the inner surface of the cell plasma membrane, and are retarded in their release from the infected cell.

Animals↗
PubMed1993

Possible involvement of microtubule disruption in bipolar budding of a Sendai virus mutant, F1-R, in epithelial MDCK cells.

Envelope glycoproteins F and HN of wild-type Sendai virus are transported to the apical plasma membrane domain of polarized epithelial MDCK cells, where budding of progeny virus occurs. On the other hand, a pantropic mutant, F1-R, buds bipolarly at both the apical and basolateral domains, and the viral glycoproteins have also been shown to be transported to both of these domains (M. Tashiro, M. Yamakawa, K. Tobita, H.-D. Klenk, R. Rott, and J.T. Seto, J. Virol. 64:4672-4677, 1990). MDCK cells were infected with wild-type virus and treated with the microtubule-depolymerizing drugs colchicine and nocodazole. Budding of the virus and surface expression of the glycoproteins were found to occur in a nonpolarized fashion similar to that found in cells infected with F1-R. In uninfected cells, the drugs were shown to interfere with apical transport of a secretory cellular glycoprotein, gp80, and basolateral uptake of [35S]methionine as well as to disrupt microtubule structure, indicating that cellular polarity of MDCK cells depends on the presence of intact microtubules. Infection by the F1-R mutant partially affected the transport of gp80, uptake of [35S]methionine, and the microtubule network, whereas wild-type virus had a marginal effect. These results suggest that apical transport of the glycoproteins of wild-type Sendai virus in MDCK cells depends on intact microtubules and that bipolar budding by F1-R is possibly due, at least in part, to the disruption of microtubules. Nucleotide sequence analyses of the viral genes suggest that the mutated M protein of F1-R might be involved in the alteration of microtubules.

Animals↗
PubMed1986

Calcium-sensitive cls4 mutant of Saccharomyces cerevisiae with a defect in bud formation.

A calcium-sensitive cls4 mutant of Saccharomyces cerevisiae ceased dividing in the presence of 100 mM CaCl2, producing large, round, unbudded cells. Since its DNA replication and nuclear division still continued after interruption of normal budding, the cls4 mutant had a defect in bud formation in Ca2+-rich medium. Its calcium content and calcium uptake activity were the same as those of the wild-type strain, suggesting that the primary defect of the mutation was not in a Ca2+ transport system. Genetic analysis showed that the cls4 mutation did not complement the cdc24-1 mutation, which is known to be a temperature-sensitive mutation affecting bud formation and localized cell surface growth at a restrictive temperature. Moreover, cls4 was tightly linked to cdc24, and a yeast 3.4-kilobase-pair DNA fragment carrying both the CLS4 and CDC24 genes was obtained. These results suggest that the cls4 mutation is allelic to the cdc24 mutation. Thus, Ca2+ ion seems to control bud formation and bud-localized cell surface growth.

Biological Transport↗
PubMed1992

SCM4, a gene that suppresses mutant cdc4 function in budding yeast.

The gene SCM4 encodes a protein which suppresses a temperature-sensitive allele of the cell division cycle gene CDC4 in Saccharomyces cerevisiae. SCM4 was cloned on a 1.8 kb BamHI fragment of yeast genomic DNA in the high copy-number vector pJDB207, which results in a 50- to 100-fold increase in the level of the 700 nucleotide SCM4 transcript in vivo. The SCM4 gene encodes a 20.2 kDa protein of 187 amino-acids with a clear tripartite domain structure in which a region rich in charged residues separates two domains of largely uncharged amino acids. Although the apparent allele specificity of cdc4 suppression suggests that the CDC4 and SCM4 proteins interact, disruption of SCM4 demonstrates that the gene product is not essential for mitosis or meiosis; however, it may be a member of a family of related, functionally redundant proteins.

Alleles↗
PubMed2006

MRX protects telomeric DNA at uncapped telomeres of budding yeast cdc13-1 mutants.

MRX, an evolutionally conserved DNA damage response complex composed of Mre11, Rad50 and Xrs2, is involved in DNA double strand break (DSB) repair, checkpoint activation and telomere maintenance. At DSBs, MRX plays a role in generating single stranded DNA (ssDNA) and signalling cell cycle arrest. Here we investigated whether MRX also contributes to generating ssDNA or signalling cell cycle arrest at uncapped telomeres. To investigate the role of MRX, we generated a conditionally degradable Rad50 protein and combined this with cdc13-1, a temperature sensitive mutation in the Cdc13 telomere capping protein. We show that Rad50 does not contribute to ssDNA generation or cell cycle arrest in response to cdcl3-1 uncapped telomeres. Instead, we find that Rad50 inhibits ssDNA accumulation and promotes cdc13-1 cell viability, consistent with a major role for MRX in telomere capping.

Base Sequence↗
PubMed2003

Use of a genome-wide approach to identify new genes that control resistance of Saccharomyces cerevisiae to ionizing radiation.

We have used the recently completed set of all homozygous diploid deletion mutants in budding yeast, S. cerevisiae, to screen for new mutants conferring sensitivity to ionizing radiation. In each strain a different open reading frame (ORF) has been replaced with a cassette containing unique 20-mer sequences that allow the relative abundance of each strain in a pool to be determined by hybridization to a high-density oligonucleotide array. Putative radiation-sensitive mutants were identified as having a reduced abundance in the pool of 4,627 individual deletion strains after irradiation. Of the top 33 strains most sensitive to radiation in this assay, 14 contained genes known to be involved in DNA repair. Eight of the remaining deletion mutants were studied. Only one, which deleted for the ORF YDR014W (which we name RAD61), conferred reproducible radiation sensitivity in both the haploid and diploid deletions and had no problem with spore viability when the haploid was backcrossed to wild-type. The rest showed only marginal sensitivity as haploids, and many had problems with spore viability when backcrossed, suggesting the presence of gross aneuploidy or polyploidy in strains initially presumed haploid. Our results emphasize that secondary mutations or deviations from euploidy can be a problem in screening this resource for sensitivity to ionizing radiation.

Blotting, Western↗
PubMed1996

Renal agenesis and hypodysplasia in ret-k- mutant mice result from defects in ureteric bud development.

The c-ret gene encodes a receptor tyrosine kinase that is expressed in the Wolffian duct and ureteric bud of the developing excretory system. Newborn mice homozygous for a mutation in c-ret displayed renal agenesis or severe hypodysplasia, suggesting a critical role for this gene in metanephric kidney development. To investigate the embryological basis of these defects, we characterized the early development of the excretory system in mutant homozygotes, and observed a range of defects in the formation, growth and branching of the ureteric bud, which account for the spectrum of renal defects seen at birth. Co-culture of isolated ureteric buds and metanephric mesenchyme show that the primary defect is intrinsic to the ureteric bud. While the mutant bud failed to respond to induction by wild-type mesenchyme, mutant mesenchyme was competent to induce the growth and branching of the wild-type bud. Furthermore, the mutant metanephric mesenchyme displayed a normal capacity to differentiate into nephric tubules when co-cultured with embryonic spinal cord. These findings suggest a model in which c-ret encodes the receptor for a (yet to be identified) factor produced by the metanephric mesenchyme, which mediates the inductive effects of this tissue upon the ureteric bud. This factor appears to stimulate the initial evagination of the ureteric bud from the Wolffian duct, as well as its subsequent growth and branching.

Animals↗
PubMed1975

Morphological and physical properties of a multiploid-forming mutant of Western equine encephalitis virus.

Morphological and physical properties of a multiploid-forming mutant of Western equine encephalitis virus were studied. Electron micrographs of the infected cells showed that most of mutant virions bud from the plasma or vacuolar membrane as a multiploid particle containing a various number of nucleocapsids enclosed with a defined common envelope. The mutant virions contained three polypeptides which migrated to the position identical with those of wild type on discontinuous acrylamide gels. Cells infected with the mutant virus synthesized the same intracellular viral RNA species as was made after infection of wild type. Cytoplasmic nucleocapsids of the mutant sedimented at 140S and contained 42S virion RNA as those of wild type; they were indistinguishable from those of wild type in an electron microscope examination. On the other hand, mutant nucleocapsids isolated from extracellular virions sedimented as heterogeneous particles larger thant 140S and were shown to be pleomorphic and aggregate in electron micrographs. The budding process of this mutant seemed to be modified, so that it might form the multiploid with the alteration of its nucleocapsids.

Animals↗
PubMed2004

SMC6 is required for MMS-induced interchromosomal and sister chromatid recombinations in Saccharomyces cerevisiae.

SMC6 (RHC18) in Saccharomyces cerevisiae, which is a homologue of the Schizosaccharomyces pombe rad18+ gene and essential for cell viability, encodes a structural maintenance of chromosomes (SMC) family protein. In contrast to the rest of the SMC family of proteins, Smc1-Smc4, which are the components of cohesin or condensin, little is known about Smc6. In this study, we generated temperature sensitive (ts) smc6 mutants of budding yeast and characterized their properties. One ts-mutant, smc6-56, ceased growth soon after up-shift to a non-permissive temperature, arrested in the late S and G2/M phase, and gradually lost viability. smc6-56 cells at a permissive temperature showed a higher sensitivity than wild-type cells to various DNA damaging agents including methyl methanesulfonate (MMS). The rad52 smc6-56 double mutant showed a sensitivity to MMS similar to that of the rad52 single mutant, indicating that Smc6 is involved in a pathway that requires Rad52 to function. Moreover, no induction of interchromosomal recombination and sister chromatid recombination was observed in smc6-56 cells, which occurred in wild-type cells upon exposure to MMS.

Base Sequence↗
PubMed2000

Mutational and hyperexpression-induced disruption of bipolar budding in yeast.

Analysis of bud-site selection in the yeast Saccharomyces cerevisiae has helped to identify many genes that are generally important for eukaryotic cell polarization. Colony morphology screens were used to identify factors relevant to the process of bipolar budding in yeast. Mutants defective in bipolar budding were identified by virtue of their inability to grow as pseudohyphae in a haploid bud3 background. A mutant allele of the MYO2 gene, encoding a class-V unconventional myosin was identified that perturbs bipolar budding without affecting axial budding and without grossly affecting the role of Myo2p in secretion and maintenance of the actin cytoskeleton. Several genes were also identified whose products, when overexpressed, are capable of disrupting bipolar budding. Among these are the actin-monomer-binding protein profilin and the Aip3p/Bud6p-interacting protein Atc1p. The results strongly support involvement of the actin cytoskeleton in the establishment of bipolar budding and in the maintenance of pseudohyphal growth.

Alleles↗
PubMed1997

Defects of urogenital development in mice lacking Emx2.

The homeobox gene Emx2 is a mouse homologue of a Drosophila head gap gene empty spiracles (ems) and is essential for the development of dorsal telencephalon (Yoshida, M., Suda, Y., Matsuo, I., Miyamoto, N., Takeda, N., Kuratani, S. and Aizawa, S. (1997) Development 124, 101-111). At the same time, Emx2 is expressed in the epithelial components of the developing urogenital system and, in Emx2 mutant mice, the kidneys, ureters, gonads and genital tracts were completely missing. Pax-2 and c-ret expressions in the Wolffian duct and WT-1 and GDNF expressions in the metanephric blastema were initially normal in the mutant. The ureteric bud grew and invaded the metanephric mesenchyme where Pax-2 expression was normally induced. Subsequently, however, Pax-2, c-ret and Lim1 expressions in the ureteric bud and GDNF expression in the mesenchyme were greatly reduced. Wnt-4 expression was never found in the mesenchyme. The tip of the ureteric bud never dilated and branching of the bud did not occur. Neither pretubular cell aggregates nor epithelialization were found in the mesenchyme. Instead the ureteric bud soon degenerated and apoptotic figures were prominent in mesenchymal cells. In explant culture, the mutant ureteric bud did not induce the epithelial transformation of the wild-type mesenchyme, and branching of the mutant ureteric bud was not induced by wild-type mesenchyme. In contrast, defects were not apparent in the mutant mesenchyme by co-culture with wild-type ureteric bud or spinal cord. These results suggest that, in metanephrogenesis, Emx2 is essential for the ureteric bud functions subsequent to Pax-2 induction in the metanephric mesenchyme. Degeneration of the Wolffian duct and mesonephric tubules was also abnormally accelerated without the formation of the Mullerian duct.

Animals↗
PubMed1998

[Role of PHO85 gene in the cell cycle control of budding yeast].

By homo-recombination with yeast intergrating plasmids, a serial of haploid mutants of budding yeast YPH499 had been constructed, which included pho85 delta strain YPH600, pho85 delta cln1 delta strain YPH610, cln1 delta cln2 delta strain YPH640 and galactose inducible strain YPH630 (pho85 delta cln1 delta cln2 delta (GAL1-10PHO85)). By analyzing the growth rate of different strains, we concluded that PHO85 gene have greater influence than CLN1 and CLN2 genes on cell growth control. After transferred from galactose media to glucose media, the tri-mutant cells collected at intervals were observed with microscope and analyzed by FACS. The results showed that the tri-mutant cells arrest in G1 phase when they were transferred to glucose media.

Cell Cycle↗
PubMed1996

A Sendai virus vector leading to the efficient expression of mutant M proteins interfering with virus particle budding.

A Sendai virus expression vector in the form of a transcribing copy-back defective interfering RNA was constructed and shown to efficiently express a tagged matrix protein in the only context of a Sendai virus infection. In an attempt to identify relevant M protein domains involved in viral assembly and budding, a series of deletion mutants were tested for their ability to bind to cellular membrane fractions. The deletion of a region spanning amino acids 105-137 significantly decreased this binding when the protein was expressed in a system driven by the T7 RNA polymerase away from any other viral proteins. Plus or minus charges were introduced in the hydrophobic portion of a predicted amphiphilic helix in this region, and M proteins with altered membrane binding properties were produced. The genes encoding these mutant M proteins were then inserted in the Sendai virus vector and shown to be expressed at levels similar to that of the endogenous wild-type M protein. The presence of a negative charge in the hydrophobic region of the putative amphiphilic helix prevented the incorporation of the mutant protein into virus particles and appeared to decrease the efficiency of virus particle budding. In contrast, the introduction of a positive charge appeared to increase the M mutant uptake into virions. The use a Sendai virus vector has therefore been shown instrumental in the identification of mutant M proteins interfering with the viral assembly-budding process.

Animals↗