Isolation, characterization, and expression analysis of zebrafish large Mafs

Large Maf proteins, which are members of the basic leucine zipper (b-Zip) superfamily, are involved in the determination and control of cellular differentiation. The expression patterns of various vertebrate large Maf mRNAs were described previously. Here, we report the cloning of a novel zebrafish large Maf cDNA, SMaf1 (Somite Maf1), and other zebrafish large Mafs, the N-terminus domains of which possess transactivational activity. We also analyzed the expression patterns of SMaf1 and SMaf2 (Somite Maf2)/Krml2 as well as MafB/Val and c-Maf during zebrafish embryogenesis. In particular, the robust expression of the novel SMaf1 mRNA, which overlapped that of MyoD, in somitic cells during somitogenesis was noteworthy. In addition, the expression patterns of SMaf2 and MafB in the blood-forming regions, and those of c-Maf and MafB in the lens cells showed spatial redundancy, although the temporal appearance of these genes at these sites differed. These data indicate that SMafs may play important roles in somitogenesis, and that Maf proteins may have overlapping and yet specific functions as to the determination and differentiation of cell lineages.     

Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon

In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSF^ts1. The mutations that resulted in a temperature-sensitive phenotype in mSF^ts1 were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42 °C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 h of growth. In order to facilitate its utilization, the mSF^ts1 was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSF^ts1 for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research.     

Origin and shaping of the laterality organ in zebrafish

Handedness of the vertebrate body plan critically depends on transient embryonic structures/organs that generate cilia-dependent leftward fluid flow within constrained extracellular environments. Although the function of ciliated organs in laterality determination has been extensively studied, how they are formed during embryogenesis is still poorly understood. Here we show that Kupffer's vesicle (KV), the zebrafish organ of laterality, arises from a surface epithelium previously thought to adopt exclusively extra-embryonic fates. Live multi-photon confocal imaging reveals that surface epithelial cells undergo Nodal/TGFbeta signalling-dependent ingression at the dorsal germ ring margin prior to gastrulation, to give rise to dorsal forerunner cells (DFCs), the precursors of KV. DFCs then migrate attached to the overlying surface epithelium and rearrange into rosette-like epithelial structures at the end of gastrulation. During early somitogenesis, these epithelial rosettes coalesce into a single rosette that differentiates into the KV with a ciliated lumen at its apical centre. Our results provide novel insights into the morphogenetic transformations that shape the laterality organ in zebrafish and suggest a conserved progenitor role of the surface epithelium during laterality organ formation in vertebrates.     

hecate, a zebrafish maternal effect gene, affects dorsal organizer induction and intracellular calcium transient frequency

A zebrafish maternal effect mutation, in the gene hecate, results in embryos that have defects in the formation of dorsoanterior structures and altered calcium release. hecate mutant embryos lack nuclear accumulation of beta-catenin and have reduced expression of genes specific to the dorsal organizer. We found that hecate mutant embryos exhibit a nearly 10-fold increase in the frequency of intracellular Ca2+ transients normally present in the enveloping layer during the blastula stages. Inhibition of Ca2+ release leads to ectopic expression of dorsal genes in mutant embryos suggesting that Ca2+ transients are important in mediating dorsal gene expression. Inhibition of Ca2+ release also results in the expression of dorsal-specific genes in the enveloping layer in a beta-catenin-independent manner, which suggests an additional function for the Ca2+ transients in this cellular layer. The mutant phenotype can be reversed by the expression of factors that activate Wnt/beta-catenin signaling, suggesting that the Wnt/beta-catenin pathway, at least as activated by an exogenous Wnt ligand, is intact in hec mutant embryos. Our results are consistent with a role for the hecate gene in the regulation of Ca2+ release during the cleavage stages, which in turn influences dorsal gene expression in both marginal cells along the dorsoventral axis and in the enveloping layer.     

Isolation and characterization of a genetic variant of bovine proinsulin

A genetic variant of bovine proinsulin has been isolated using preparative reverse-phase HPLC. The new proinsulin (bovine proinsulin II) differs from the known proinsulin (bovine proinsulin I) by a single amino acid residue at position C-48 in the connecting peptide. The amino acid replacement is a leucine substitution for proline. The two proinsulins were found in a ratio of approximately 9:1, proinsulin I: proinsulin II. No chemical or biological differences were observed for the two proinsulins other than their different elution times on reverse-phase HPLC.     

Isolation and characterization of herC, a mutation of Escherichia coli affecting maintenance of ColE1

Summary Two modes of ColE1 DNA replication are known, one dependent on RNase H, and the other RNase H independent. The cer114 mutant of the ColE1 replicon is defective in both modes and carries a single base pair alteration 95 by upstream of the replication origin. An Escherichia coli mutant which restored maintenance of the cer114 replicon was isolated. This host suppressor mutant is defective in RNase H and carries a herC, mutation located at 62 min of the E. coli chromosome. The herC, mutation is recessive to its wild-type allele and supports maintenance of the mutant replicon in the absence of RNase H. The herC, mutation alone conferred cold-sensitive growth, suggesting that the herC, gene product is essential for cell growth. The 1832 by E. coli DNA fragment, containing the wild-type allele of the herC, mutation, was cloned and an open reading frame for the HerC protein was determined.     

fsi zebrafish show concordant reversal of laterality of viscera, neuroanatomy, and a subset of behavioral responses

Asymmetries in CNS neuroanatomy are assumed to underlie the widespread cognitive and behavioral asymmetries in vertebrates. Studies in humans have shown that the laterality of some cognitive asymmetries is independent of the laterality of the viscera; discrete mechanisms may therefore regulate visceral and neural lateralization. However, through analysis of visceral, neuroanatomical, and behavioral asymmetries in the frequent-situs-inversus (fsi) line of zebrafish, we show that the principal left-right body asymmetries are coupled to certain brain asymmetries and lateralized behaviors. fsi fish with asymmetry defects show concordant reversal of heart, gut, and neuroanatomical asymmetries in the diencephalon. Moreover, the neuroanatomical reversals in reversed fsi fish correlate with reversal of some behavioral responses in both fry and adult fsi fish. Surprisingly, two behavioral asymmetries do not reverse, suggesting that at least two separable mechanisms must influence functional lateralization in the CNS. Partial reversal of CNS asymmetries may generate new behavioral phenotypes; supporting this idea, reversed fsi fry differ markedly from their normally lateralized siblings in their behavioral response to a novel visual feature. Revealing a link between visceral and brain asymmetry and lateralized behavior, our studies help to explain the complexity of the relationship between the lateralities of visceral and neural asymmetries.     

Isolation and characterization of OLE1, a gene affecting fatty acid desaturation from Saccharomyces cerevisiae

The unsaturated fatty acid (ufa) requiring ole1 mutant of Saccharomyces cerevisiae appears to produce a defective delta-9 fatty acid desaturase. This enzyme catalyzes double bond formation between carbons 9 and 10 of palmitoyl and stearoyl coenzyme A. A DNA fragment isolated by complementation of an ole1 strain repairs the ufa requirement in mutant cells. Genetic analysis of the cloned DNA fragment indicates that it is allelic to the OLE1 gene. Disruption of a single copy of the wild type gene in a diploid strain produces both wild type and nonreverting ufa-requiring haploid progeny upon sporulation. Membrane lipids of the disrupted haploid strains contain only ufas supplied in the growth medium. The recovery of activity in both wild type and disrupted segregants was examined after removal of ufas from the growth medium. Following ufa deprivation disruptant cells grew normally for about three generations and then at a slower rate for at least 0.6 generations. During that time cellular ufas dropped from 63 to 7.3 mol % of the total fatty acids. No production of the 16:1 and 18:1 products of the desaturase was observed in disruptant cells, whereas desaturation in wild type control cells was evident 2 h after deprivation. These results indicate that 1) the OLE1 gene is essential for production of monounsaturated fatty acids and is probably the structural gene for the delta-9 desaturase enzyme. 2) A large part of membrane ufas present under normal culture conditions are not essential for growth and cell division.     

Characterization of a weak allele of zebrafish cloche mutant

Hematopoiesis is a complicated and dynamic process about which the molecular mechanisms remain poorly understood. Danio rerio (zebrafish) is an excellent vertebrate system for studying hematopoiesis and developmental mechanisms. In the previous study, we isolated and identified a cloche(172) (clo(172)) mutant, a novel allele compared to the original cloche (clo) mutant, through using complementation test and initial mapping. Here, according to whole mount in-situ hybridization, we report that the endothelial cells in clo(172) mutant embryos, although initially developed, failed to form the functional vascular system eventually. In addition, further characterization indicates that the clo(172) mutant exhibited weaker defects instead of completely lost in primitive erythroid cells and definitive hematopoietic cells compared with the clo(s5) mutant. In contrast, primitive myeloid cells were totally lost in clo(172) mutant. Furthermore, these reappeared definitive myeloid cells were demonstrated to initiate from the remaining hematopoietic stem cells (HSCs) in clo(172) mutant, confirmed by the dramatic decrease of lyc in clo(172)runx1(w84x) double mutant. Collectively, the clo(172) mutant is a weak allele compared to the clo(s5) mutant, therefore providing a model for studying the early development of hematopoietic and vascular system, as well as an opportunity to further understand the function of the cloche gene.     

The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP⁺-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex ¹, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex ¹ mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP⁺-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex ¹ mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex ¹ and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development. This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to M.M.B.     

Isolation and characterization of mutations affecting UDPG pyrophosphorylase activity in Dictyostelium discoideum

A procedure for screening large numbers of clones for an enzyme activity was used to isolate mutations which affect UDPG pyrophosphorylase activity (EC 2.7.7.9) in the cellular slime mold Dictyostelium discoideum. Five strains were recovered which have little or no UDPG pyrophosphorylase activity. Ten other strains were found which have significant activity in vivo which is rapidly inactivated upon cell lysis. These strains have permitted us to evaluate the role of UDPG pyrophosphorylase during growth and development. The enzyme affects the growth rate of the cells but is not essential for growth. However, during development the lack of enzyme activity leads to cell death and lysis. Strains which lack UDPG pyrophosphorylase accomplish early developmental events but are unable to culminate. However, certain biochemical and cytological differentiations associated with late stages were observed.     

Isolation, characterization, and genetic analysis of monosomic, aneuploid mutants of Candida albicans

A white, prototrophic Candida albicans strain, heterozygous for the ADE2 gene (ade2/ADE2), was treated with the antimitotic agent methyl benzimidazole carbamate, and yielded red, adenine-requiring colonies at a rate of 4 x 10(-3), an order of magnitude higher than the spontaneous rate of Ade- colony formation. These red Ade- colonies were small, growing at approximately half the rate of the parent strain, and gave rise to large red colonies spontaneously. When the chromosomes of the small red colonies were separated by pulsed-field gel electrophoresis, the band hybridizing with the ADE2 gene was diminished in staining intensity by half relative to the parent and large red-colony strains. Restriction fragment-length polymorphism analysis and auxotrophic mutant spectra after mutagenesis suggested that the small red Ade- strains were monosomic aneuploids lacking one of a pair of chromosome homologues, while the large red strains had regained a homologue, presumably via a second non-disjunction event. Parasexual genetic analysis of two of the auxotrophs isolated from a putative aneuploid suggested that both mutations were linked to the ADE2 gene. These experiments suggest that targeted chromosome loss and monosomic, aneuploid strains have the potential to extend the scope of genetic analysis in this diploid, asexual organism.     

Isolation and genetic characterization of ethanol-resistant reovirus mutants.

To better understand the mechanism(s) by which viruses respond to chemical or physical treatments, we isolated a series of mutant strains of reovirus type 3 Dearing that exhibit increased ethanol resistance. Following exposure to 33% ethanol for 20 min, the parental strain exhibited a 5 log10 decrease in infectivity. The mutant strains, however, exhibited a 2 to 3 log10 decrease in titer following identical treatment. Through the use of reassortant viruses, we mapped this increased ethanol resistance mutation to the M2 gene segment, which encodes a major outer capsid protein, mu1C. Sequence analysis of mutant M2 genes revealed that six of seven unique mutants possessed single-point mutations in this gene. In addition, the change in six of seven mutants caused a predicted amino acid change in a 35-amino-acid region of the gene product between amino acids 425 and 459. The identification of ethanol resistance mutations within a discrete region of this outer capsid protein identifies that portion of the protein as important in reovirus stability. The presence of viral particles possessing altered stability also suggests that subpopulations of viruses may possess altered environmental stability, which, in turn, could affect viral transmission.     

Isolation and characterization of a transposon mutant of Pseudomonas aeruginosa affecting uptake of dibenzothiophene in n-tetradecane

AIMS: Isolation and characterization of a transposon mutant of Pseudomonas aeruginosa affecting the uptake of dibenzothiophene (DBT) in n-tetradecane (n-TD). METHODS AND RESULTS: The dsz desulphurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred to the chromosome of P. aeruginosa NCIMB9571 using a transposon vector. A recombinant (named PARM1) was obtained which was able to desulphurize DBT in water, but not in n-TD. CONCLUSIONS: PARM1 is a mutant deficient in a DBT transport system operational in n-TD. This transport system is independent of rhamnolipids and of the n-alkane transport system. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas aeruginosa NCIMB9571 seems to have a specific system of transporting hydrophobic compounds such as DBT in oil.     

Isolation,genetic and functional characterization of novel soil nirK-type denitrifiers

Denitrification, the reduction of nitrogen oxides (NO₃⁻ and NO₂⁻) to N₂ via the intermediates NO and N₂O, is crucial for nitrogen turnover in soils. Cultivation-independent approaches that applied nitrite reductase genes (nirK/nirS) as marker genes to detect denitrifiers showed a predominance of genes presumably derived from as yet uncultured organisms. However, the phylogenetic affiliation of these organisms remains unresolved since the ability to denitrify is widespread among phylogenetically unrelated organisms. In this study, denitrifiers were cultured using a strategy to generally enrich soil microorganisms. Of 490 colonies screened, eight nirK-containing isolates were phylogenetically identified (16S rRNA genes) as members of the Rhizobiales. A nirK gene related to a large cluster of sequences from uncultured bacteria mainly retrieved from soil was found in three isolates classified as Bradyrhizobium sp. Additional isolates were classified as Bradyrhizobium japonicum and Bosea sp. that contained nirK genes also closely related to the nirK from these strains. These isolates denitrified, albeit with different efficiencies. In Devosia sp., nirK was the only denitrification gene detected. Two Mesorhizobium sp. isolates contained a nirK gene also related to nirK from cultured Mesorhizobia and uncultured soil bacteria but no gene encoding nitric oxide or nitrous oxide reductase. These isolates accumulated NO under nitrate-reducing conditions without growth, presumably due to the lethal effects of NO. This showed the presence of a functional nitrite reductase but lack of a nitric oxide reductase. In summary, similar nirK genotypes recurrently detected mainly in soils likely originated from Rhizobia, and functional differences were presumably strain-dependent.