Split-ubiquitin yeast two-hybrid interaction reveals a novel interaction between a natural resistance associated macrophage protein and a membrane bound thioredoxin in Brassica juncea

Natural resistance associated macrophage proteins (NRAMPs) are evolutionarily conserved metal transporters involved in the transport of essential and nonessential metals in plants. Fifty protein interactors of a Brassica juncea NRAMP protein was identified by a Split-Ubiquitin Yeast-Two-Hybrid screen. The interactors were predicted to function as components of stress response, signaling, development, RNA binding and processing. BjNRAMP4.1 interactors were particularly enriched in proteins taking part in photosynthetic or light regulated processes, or proteins predicted to be localized in plastid/chloroplast. Further, many interactors also had a suggested role in cellular redox regulation. Among these, the interaction of a photosynthesis-related thioredoxin, homologous to Arabidopsis HCF164 (High-chlorophyll fluorescence164) was studied in detail. Homology modeling of BjNRAMP4.1 suggested that it could be redox regulated by BjHCF164. In yeast, the interaction between the two proteins was found to increase in response to metal deficiency; Mn excess and exogenous thiol. Excess Mn also increased the interaction in planta and led to greater accumulation of the complex at the root apoplast. Network analysis of Arabidopsis homologs of BjNRAMP4.1 interactors showed enrichment of many protein components, central to chloroplastic/cellular ROS signaling. BjNRAMP4.1 interacted with BjHCF164 at the root membrane and also in the chloroplast in accordance with its proposed function related to photosynthesis, indicating that this interaction occurred at different sub-cellular locations depending on the tissue. This may serve as a link between metal homeostasis and chloroplastic/cellular ROS through protein–protein interaction.     

Cloning of Flagellar Genes in Chlamydomonas Reinhardtii by DNA Insertional Mutagenesis

Chlamydomonas is a popular genetic model system for studying many cellular processes. In this report, we describe a new approach to isolate Chlamydomonas genes using the cloned nitrate reductase gene (NIT1) as an insertional mutagen. A linearized plasmid containing the NIT1 gene was introduced into nit1 mutant cells by glass-bead transformation. Of 3000 Nit(+) transformants examined, 74 showed motility defects of a wide range of phenotypes, suggesting that DNA transformation is an effective method for mutagenizing cells. For 13 of 15 such motility mutants backcrossed to nit(-) mutant strains, the motility phenotype cosegregated with the Nit(+) phenotype, indicating that the motility defects of these 13 mutants may be caused by integration of the plasmid. Further genetic analysis indicated that three of these mutants contained alleles of previously identified loci: mbo2 (move backward only), pf13 (paralyzed flagella) and vfl1 (variable flagellar number). Three other abnormal-flagellar-number mutants did not map to any previously described loci at which mutations produce similar phenotypes. Genomic sequences flanking the integrated plasmid in the mbo2 and vfl1 mutants were isolated and used as probes to obtain wild-type genomic clones, which complemented the motility defects upon transformation into cells. Our results demonstrate the potential of this new approach for cloning genes identified by mutation in Chlamydomonas.     

The hindsight gene is required for epithelial maintenance and differentiation of the tracheal system in Drosophila

During animal development, morphogenesis of tissues and organs requires dynamic cell shape changes and movements that are accomplished without loss of epithelial integrity. Data from vertebrate and invertebrate systems have implicated several cell surface and cytoskeleton-associated molecules in the establishment and maintenance of epithelial architecture, but there has been little analysis of the genetic regulatory hierarchies that control epithelial morphogenesis in specific tissues. Here we show that the Drosophila Hindsight nuclear zinc-finger protein is required during tracheal morphogenesis for the maintenance of epithelial integrity and assembly of apical extracellular structures known as taenidia. In hindsight (hnt) mutants tracheal placodes form, invaginate, and undergo primary branching as well as early fusion events. Starting at midembryogenesis, however, the tracheal epithelium collapses or expands to give rise to sacs of tissue. While a subset of hnt mutant tracheal cells enters the apoptotic pathway, genetic suppression of apoptosis indicates that this is not the cause of the epithelial defects. Surviving hnt mutant tracheal cells retain cell-cell junctions and a normal subcellular distribution of apical markers such as Crumbs and DE-Cadherin. However, taenidia do not form on the lumenal surface of tracheal cells. While loss of epithelial integrity is a common feature of crumbs, stardust, and hnt mutants, defective assembly of taenidia is unique to hnt mutants. These data suggest that HNT is a tissue-specific factor that regulates maintenance of the tracheal epithelium as well as differentiation of taenidia.     

Molecular Genetic Analysis of the DILUTE-SHORT EAR ( d-se) Region of the Mouse

Genes of the dilute-short ear (d-se) region of mouse chromosome 9 comprise an array of loci important to the normal development of the animal. Over 200 spontaneous, chemically induced and radiation-induced mutations at these loci have been identified, making it one of the most genetically well-characterized regions of the mouse. Molecular analysis of this region has recently become feasible by the identification of a dilute mutation that was induced by integration of an ecotropic murine leukemia virus genome. Several unique sequence cellular DNA probes flanking this provirus have now been identified and used to investigate the organization of wild-type chromosomes and chromosomes with radiation-induced d-se region mutations. As expected, several of these mutations are associated with deletions, and, in general, the molecular and genetic complementation maps of these mutants are concordant. Furthermore, a deletion breakpoint fusion fragment has been identified and has been used to orient the physical map of the d-se region with respect to the genetic complementation map. These experiments provide important initial steps for analyzing this developmentally important region at the molecular level, as well as for studying in detail how a diverse group of mutagens acts on the mammalian germline.     

Repair and regeneration: opportunities for carcinogenesis from tissue stem cells

This review will discuss the mechanisms of repair and regeneration in various tissue types and how dysregulation of these mechanisms may lead to cancer. Normal tissue homeostasis involves a careful balance between cell loss and cell renewal. Stem and progenitor cells perform these biologic processes as the functional units of regeneration during both tissue homeostasis and repair. The concept of tissue stem cells capable of giving rise to all differentiated cells within a given tissue led to the concept of a cellular hierarchy in tissues and in tumors. Thus, only a few cells may be necessary and sufficient for tissue repair or tumor regeneration. This is known as the hierarchical model of tumorigenesis. This report will compare this model with the stochastic model of tumorigenesis. Under normal circumstances, the processes of tissue regeneration or homeostasis are tightly regulated by several morphogen pathways to prevent excessive or inappropriate cell growth. This review presents the recent evidence that dysregulation of these processes may provide opportunities for carcinogenesis for the long-lived, highly proliferative tissue stem cell population. New findings of cancer initiating tissue stem cells identified in several solid and circulating cancers including breast, brain and hematopoietic tumors will also be reviewed. Finally, this report reviews the cellular biology of cancer and its relevance to the development of more effective cancer treatment protocols.     

Genetic Interactions between Naturally Occurring Alleles at Quantitative Trait Loci and Mutant Alleles at Candidate Loci Affecting Bristle Number in Drosophila Melanogaster

Previously, we mapped quantitative trait loci (QTL) affecting response to short-term selection for abdominal bristle number to seven suggestive regions that contain loci involved in bristle development and/or that have adult bristle number mutant phenotypes, and are thus candidates for bristle number QTL in natural populations. To test the hypothesis that the factors contributing to selection response genetically interact with these candidate loci, high and low chromosomes from selection lines were crossed to chromosomes containing wild-type or mutant alleles at the candidate loci, and the numbers of bristles were recorded in trans heterozygotes. Quantitative failure to complement, detected as a significant selection line*cross effect by analysis of variance, can be interpreted as evidence for allelism or epistasis between the factors on selected chromosomes and the candidate loci. Mutations at some candidate loci (bb, emc, h, Dl, Hairless) showed strong interactions with selected chromosomes, whereas others interacted weakly (ASC, abd, Scr) or not at all (N, mab, E(spl)). These results support the hypothesis that some candidate loci, initially identified through mutations of large effect on bristle number, either harbor or are close members in the same genetic pathway as variants that contribute to standing variation in bristle number.     

The R2TP complex: discovery and functions

The two closely related AAA+family ATPases Rvb1 and Rvb2 are part of several critical multiprotein complexes, and, thus, are involved in a wide range of cellular processes including chromatin remodelling, telomerase assembly, and snoRNP biogenesis. It was found that Rvb1 and Rvb2 form a tight functional complex with Pih1 (Protein interacting with Hsp90) and Tah1 (TPR-containing protein associated with Hsp90), which are two Hsp90 interactors. We named the complex R2TP. The complex was originally isolated from Saccharomyces cerevisiae and was, subsequently, identified in mammalian cells. R2TP was found to be required for box C/D snoRNP biogenesis in yeast and mammalian cells. More recently, several studies revealed that the complex is also involved in multiple biological processes including apoptosis, phosphatidylinositol-3 kinase-related protein kinase (PIKK) signalling, and RNA polymerase II assembly. In this review, we describe the discovery of the complex and discuss the emerging critical roles that R2TP plays in distinct cellular processes.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Genome-wide associations between hybrid sterility QTL and marker transmission ratio distortion

Marker transmission ratio distortion (TRD) in genetic mapping populations is frequently ascribed to selection against allelic combinations that cause hybrid incompatibility. Accordingly, genomic regions of TRD should be nonrandomly associated (colocated) with loci that underlie hybrid incompatibility. To directly test this hypothesis, we evaluated the genome-wide qualitative and quantitative agreement between chromosomal regions exhibiting marker TRD and those known to contain hybrid incompatibility quantitative trait locus (QTL). Incompatibility data came from a near-isogenic line (NIL) analysis of pollen and seed sterility in a cross between two Solanum (formerly Lycopersicon) species. We assessed (1) whether these incompatibility loci are colocated with markers that show significant TRD in two earlier generations preceding these introgression lines and (2) whether the magnitude of marker distortion quantitatively matches the estimated strength of selection against each incompatibility locus. We found evidence that TRD regions are chromosomally colocated with hybrid incompatibility loci more frequently than is expected by chance: pollen sterility QTLs were most closely associated with distorted heterozygote frequencies in later-generation backcrosses. Nonetheless, there was no evidence for an association between TRD and seed sterility and little evidence of a quantitative association between the magnitude of marker TRD and the fitness effects of heterospecific alleles at each chromosomal location. We propose and test a model (the "dance partner" model) to explain several cases where regions of TRD are not associated with hybrid incompatibility loci. Under this model, some NILs containing greater than one heterospecific introgression may not express hybrid incompatibility phenotypes because they carry both appropriate genetic dance partners required for a fully functional interaction. Accordingly, negative interactions expressed in earlier backcross generations are masked in these double-introgression NILs. Based on this model, we identify the location of several new putative pairwise interactors underlying hybrid incompatibility in this species cross.     

Partial blocks in the early steps of the chlorophyll synthesis pathway: A common feature of chlorophyll b-deficient mutants

We have analyzed precursor pools in the chlorophyll (Chi) synthesis pathway for a set of eighteen well studied Chl b -defident mutants in monocotyledonous (barley, maize and wheat) and dicotyledonous plants ( Antirrhinum, Arabidopsis , soybean, tobacco and tomato) that form abnormal thylakoid membrane systems. All of these mutants have a partial block in Chl synthesis and nearly all of them accumulate protoporphyrin IX (Proto), the last porphyrin compound common to both heme and Chl synthesis. The large number of mutants at several genetic loci affecting this critical branchpoint in tetrapyrrole biosynthesis suggests that the Mg-chelatase enzyme, catalyzing the first committed step of Chi biosynthesis, is a multimeric complex composed of the products of some of these genetic loci, and perhaps regulated by others. We hypothesize that these mutants are Chi b -deficient and have reduced amounts of light-harvesting antenna complexes (LHCs.) and develop abnormal thylakoid membranes as a direct result of limited Chl synthesis. The observed bottleneck in Chl synthesis can also explain the light-intensity-dependent and temperature-dependent expression of the mutant phenotype. This hypothesis offers a simple explanation for the wide variety of pbenotypes that have been reported for the many Chl-deficient mutants in the literature. Our findings are also consistent with the notion that Chl b is made from "left over" Chl a molecules and suggest that the Chi b -deficient mutants should be considered more appropriately as leaky Chl-deficient mutants.     

Functional and phenotypic relevance of differentially expressed proteins in calcineurin mutants of Caenorhabditis elegans

Calcineurin is a heterodimeric serine/threonine protein phosphatase, important for many cellular processes such as T-cell regulation, cardiac hypertrophy and kidney development. We previously reported the characterization of Caenorhabditis elegans calcineurin mutants as providing a simple but excellent genetic model system for studying in vivo functions of calcineurin. Calcineurin loss-of-function mutants, cnb-1(lf), and gain-of-function mutants, tax-6(gf), show certain opposite phenotypes as well as some similar phenotypes. In order to explain the phenotypic similarity observed in both loss-of-function and gain-of-function mutants, we examined the proteins that followed similar trends in both mutants relative to wild-type worms by using 2-DE. Interestingly, VHA-13, HSP-6 and phosphoenolpyruvate carboxykinase are down-regulated in both mutants. A total of 96 differentially regulated proteins were identified by MALDI-TOF/MS. Among these, 42 proteins are up-regulated and 54 proteins are down-regulated in calcineurin mutants. Furthermore, knock-down of about 30% of the genes, which are down-regulated in calcineurin mutants, showed some of the phenotypes of calcineurin-null mutants. This analysis suggests the functional relevance of these proteins to calcineurin activity in C. elegans.     

Protruding vulva mutants identify novel loci and Wnt signaling factors that function during Caenorhabditis elegans vulva development

The Caenorhabditis elegans vulva develops from the progeny of three vulval precursor cells (VPCs) induced to divide and differentiate by a signal from the somatic gonad. Evolutionarily conserved Ras and Notch extracellular signaling pathways are known to function during this process. To identify novel loci acting in vulval development, we carried out a genetic screen for mutants having a protruding-vulva (Pvl) mutant phenotype. Here we report the initial genetic characterization of several novel loci: bar-1, pvl-4, pvl-5, and pvl-6. In addition, on the basis of their Pvl phenotypes, we show that the previously identified genes lin-26, mom-3/mig-14, egl-18, and sem-4 also function during vulval development. Our characterization indicates that (1) pvl-4 and pvl-5 are required for generation/survival of the VPCs; (2) bar-1, mom-3/mig-14, egl-18, and sem-4 play a role in VPC fate specification; (3) lin-26 is required for proper VPC fate execution; and (4) pvl-6 acts during vulval morphogenesis. In addition, two of these genes, bar-1 and mom-3/mig-14, are known to function in processes regulated by Wnt signaling, suggesting that a Wnt signaling pathway is acting during vulval development.     

Cytogenetic Definition and Morphogenetic Analysis of Delta, a Gene Affecting Neurogenesis in Drosophila Melanogaster

We have conducted a genetic analysis of a small interval of the third chromosome known to include Delta (Dl), a locus that affects the segregation of the ectoderm into neural and epidermal lineages during embryogenesis and the morphogenesis of some ectodermally derived structures, in Drosophila melanogaster. This analysis has led to the definition of seven independent complementation groups, one of which is Delta, within the interval extending from 91F6-13 to 92A2. Among the extant mutations in these seven loci, only mutations in Dl lead to the so-called neurogenic phenotype: hypertrophy of the nervous system and reduction of the epidermis. Combined cytogenetic and genetic analyses allow us to define absolute proximal (91F5-92A1) and distal (92A2) cytogenetic limits for the Dl locus. We have isolated hypomorphic and amorphic alleles of Dl and find that, for any given allele, there is an inverse correlation between neural hypertrophy and epidermal reduction in embryos and a direct correlation between the severity of embryonic phenotypes in mutant homozygotes and hemizygotes and the imaginal phenotype in heterozygous adults.     

Genetic analyses of Rhizobium meliloti exopolysaccharides

We have recently obtained strong genetic evidence that the acidic Calcofluor-binding exopolysaccharide (EPS I) of Rhizobium meliloti Rm1021 is required for nodule invasion and possibly for later events in nodule development. Thirteen loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. exoH mutants fail to succinylate their EPS I and form empty Fix- nodules. We have identified two unlinked regulatory loci, exoR and exoS, whose products play negative roles in the regulation of expression of the exo genes. We have recently discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for Rhizobium exopolysaccharides in nodulation are discussed.     

Genetic Analysis of Delta, a Neurogenic Gene of Drosophila melanogaster

We report here the results of a genetic analysis of the gene Delta (Dl) of Drosophila melanogaster. Dl has been mapped to the band 92A2, on the basis of two pieces of evidence: (1) this band is the common breakpoint of several chromosomal aberrations associated with Dl mutations and (2) recombination mapping of alleles of five different lethal complementation groups that are uncovered by Df( 3R)Dl(FX3) (breakpoints at 91F11; 92A3). Dl was found to map most distally of all five complementation groups. The analysis of a large number of Dl alleles demonstrates the considerable genetic and functional complexity of Dl. Three types of Dl alleles are distinguishable. Most alleles behave as amorphic or hypomorphic recessive embryonic lethal alleles, which in addition cause various defects in heterozygosity over the wild-type allele. The defects are due to haplo-insufficient expression of the locus and can be suppressed by a duplication of the wild-type allele. The second class is comprised of three alleles with antimorphic expression. The phenotype of these alleles can only be reduced, rather than suppressed, by a duplication of the wild-type allele. The third group is comprised of three visible, predominantly hypomorphic alleles with an antimorphic component of phenotypic expression. The pattern of interallelic complementation is complex. On the one hand, there is a group of hypomorphic, fully penetrant embryonic lethal alleles which complement each other. On the other hand, most alleles, including all amorphic alleles, are viable over the visible ones; alleles of antimorphic expression, however, are lethal over visible alleles. These results are compatible with a rather complex genetic organization of the Dl locus.     

Systematic Screens for Proteins That Interact with the Mucolipidosis Type IV Protein TRPML1

Mucolipidosis type IV is a lysosomal storage disorder resulting from mutations in the MCOLN1 gene, which encodes the endosomal/lysosomal Transient Receptor Potential channel protein mucolipin-1/TRPML1. Cells isolated from Mucolipidosis type IV patients and grown in vitro and in in vivo models of this disease both show several lysosome-associated defects. However, it is still unclear how TRPML1 regulates the transport steps implicated by these defects. Identifying proteins that associate with TRPML1 will facilitate the elucidation of its cellular and biochemical functions. We report here two saturation screens for proteins that interact with TRPML1: one that is based on immunoprecipitation/mass spectrometry and the other using a genetic yeast two-hybrid approach. From these screens, we identified largely non-overlapping proteins, which represent potential TRPML1-interactors., Using additional interaction assays on some of the potential interactors from each screen, we validated some proteins as candidate TRPML1 interactors In addition, our analysis indicates that each of the two screens not only identified some false-positive interactors, as expected from any screen, but also failed to uncover potential TRPML1 interactors. Future studies on the true interactors, first identified in these screens, will help elucidate the structure and function of protein complexes containing TRPML1.     

Mutations in Genes Encoding Essential Mitotic Functions in DROSOPHILA MELANOGASTER

Temperature-sensitive mutations at 15 loci that affect the fidelity of mitotic chromosome behavior have been isolated in Drosophila melanogaster. These mitotic mutants were detected in a collection of 168 EMS-induced X-linked temperature-sensitive (ts) lethal and semilethal mutants. Our screen for mutations with mitotic effects was based upon the reasoning that under semirestrictive conditions such mutations could cause an elevated frequency of mitotic chromosome misbehavior and that such events would be detectable with somatic cell genetic techniques. Males hemizygous for each ts lethal and heterozygous for the recessive autosomal cell marker mwh were reared under semirestrictive conditions, and the wings of those individuals surviving to adulthood were examined for an increased frequency of mwh clones. Those mutations producing elevated levels of chromosome instability during growth of the wing imaginal disc were also examined for their effects on chromosome behavior in the cell lineages producing the abdominal cuticle. Fifteen mutations affect chromosome behavior in both wing and abdominal cells and thus identify loci generally required for the fidelity of mitotic chromosome transmission. Mapping and complementation tests show that these mutations represent 15 loci. One mutant is an allele of a locus (mus-101) previously identified by mutagen-sensitive mutants and a second mutant is an allele of the lethal locus zw 10.--The 15 mutants were also examined cytologically for their effects on chromosomes in larval neuroblasts. Taken together, the results of our cytological and genetical studies show that these mutants identify loci with wild-type functions necessary for either maintenance of chromosome integrity or regular disjunction of chromosomes or chromosome condensation. Thus, these mutations define a broad spectrum of genes required for the normal execution of the mitotic chromosome cycle.     

Genetic analysis of tissue glutathione concentrations and redox balance

Glutathione redox balance—defined as the ratio GSH/GSSG—is a critical regulator of cellular redox state, and declines in this ratio are closely associated with oxidative stress and disease. However, little is known about the impact of genetic variation on this trait. Previous mouse studies suggest that tissue GSH/GSSG is regulated by genetic background and is therefore heritable. In this study, we measured glutathione concentrations and GSH/GSSG in liver and kidney of 30 genetically diverse inbred mouse strains. Genetic background caused an approximately threefold difference in hepatic and renal GSH/GSSG between the most disparate strains. Haplotype association mapping determined the loci associated with hepatic and renal glutathione phenotypes. We narrowed the number of significant loci by focusing on those located within protein-coding genes, which we now consider to be candidate genes for glutathione homeostasis. No candidate genes were associated with both hepatic and renal GSH/GSSG, suggesting that genetic regulation of GSH/GSSG occurs predominantly in a tissue-specific manner. This is the first quantitative trait locus study to examine the genetic regulation of glutathione concentrations and redox balance in mammals. We identified novel candidate genes that have the potential to redefine our knowledge of redox biochemistry and its regulation and inform future therapeutic applications.