Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision

Gene targeting is widely used for the precise manipulation of genes. However, in the model organism Caenorhabditis elegans non-transposon mediated gene targeting remains laborious, and as a result has not been widely used. One obstacle to the wider use of this approach is the difficulty of identifying homologous recombination events amongst non-specific events. To improve gene targeting in C. elegans, we used a counter-selection approach to reduce the number of false positives; this involved using unc-119 as a positive-selection marker and GFP as a counter-selection marker which is lost during homologous recombination. This method of gene targeting allows straightforward screening for homologous events using a dissecting microscope equipped for fluorescence. In addition, to improve the final engineered product, we utilised Flp recombinase to remove the unc-119 selection marker, in somatic cells, producing clean knockouts in these cells. Using this strategy we have produced a knockout of the plc-4 gene, which encodes phospholipase C-δ in C. elegans, and demonstrated that conditional gene knockout is feasible in C. elegans.     

Essential Genes and Deficiencies in the UNC-22 IV Region of CAENORHABDITIS ELEGANS

Five formaldehyde-induced deficiencies that uncover unc-22 IV, a gene affecting muscle structure in the nematode Caenorhabditis elegans were isolated and positioned. The largest deficiency, sDf2, extends in both directions from unc-22 and is approximately 1.0–2.0 map units in length. The other four deficiencies, sDf7, sDf8, sDf9 and sDf10, are all smaller than sDf2 and are located within the region uncovered by this deficiency. Thirty-seven ethyl methanesulfonate-induced lethal and sterile mutations linked to unc-22 were isolated and tested for complementation with sDf2. Nineteen lethal mutations failed to complement sDf2. Sixteen of these were further positioned by recombination mapping and also by deficiency mapping with sDf7, sDf8, sDf9 and sDf10. These sixteen mutations define 11 new essential genes in this region. Eight of the genes lie in a 0.9-map unit interval to the left of unc-22, whereas the three remaining genes lie in a region of about 0.2 map units to the right of unc-22. We believe that two of the essential genes identified in this study, let-56 and let-52, are the adjacent genes on either side of unc-22. The lethal mutations exhibit a wide range of terminal phenotypes: from first stage larva to sterile adult.     

Mutations in the unc-54 myosin heavy chain gene of caenorhabditis elegans that alter contractility but not muscle structure

Reversion analysis of mutants of unc-22 IV, a gene affecting muscle structure and function in Caenorhabditis elegans, led to the isolation of six extragenic dominant suppressors of the “twitching” phenotype of unc-22 mutants. All six suppressors are new alleles of unc-54 I, the major body wall myosin heavy chain gene. Homozygous suppressor strains are slow, stiff and have normal muscle structure, whereas previously identified unc-54 alleles confer flaccid paralysis and drastic reduction in thick filament number and organization. Placement of the three suppressor mutations s74, s77 and s95 on the genetic fine structure map of unc-54 demonstrates that they are clustered near the right end of the map. Since this end of the gene corresponds to the 5′ end of the coding sequence, these suppressor mutations probably result in amino acid substitutions in the globular head of the myosin molecule, and should be of value in studies of myosin force generation.     

Genetic Organization of the Region around UNC-15 (I), a Gene Affecting Paramyosin in CAENORHABDITIS ELEGANS

In the nematode Caenorhabditis elegans mutants in the gene unc-15 (I) affect the muscle protein paramyosin (Waterston, Fishpool and Brenner 1977). We have characterized 20 ethyl methanesulfonate-induced mutations in essential genes closely linked to unc-15. These lethals defined 16 new complementation groups. In the 0.65 map-unit interval around unc-15 defined by dpy-14 and unc-56, seven newly identified genes have been mapped relative to five existing genes. At present, the average distance between genes in this region is approximately 0.05 map units. Two genes, unc-15 and unc-13, are only 0.025 map units apart. Partial fine-structure maps of alleles of these two genes have been constructed. This analysis of unc-15 and genes adjacent to it is the first in a series of genetic and biochemical studies directed towards understanding the control of unc-15 expression.     

Genetic organization of the unc-22 IV gene and the adjacent region in Caenorhabditis elegans

Summary The genetic organization of the region immediately adjacent to the unc-22 IV gene in Caenorhabditis elegans has been studied. We have identified twenty essential genes in this interval of approximately 1.5-map units on Linkage Group IV. The mutations that define these genes were positioned by recombination mapping and complementation with several deficiencies. With few exceptions, the positions obtained by these two methods agreed. Eight of the twenty essential genes identified are represented by more than one allele. Three possible internal deletions of the unc-22 gene have been located by intra-genic mapping. In addition, the right end point of a deficiency or an inversion affecting the adjacent genes let-56 and unc-22 has been positioned inside the unc-22 gene.     

Spontaneous Unstable UNC-22 IV Mutations in C. ELEGANS Var. Bergerac

This paper describes a mutator system in the nematode Caenorhabditis elegans var. Bergerac for the gene unc-22. Of nine C. elegans and two C. briggsae strains tested only the Bergerac BO strain yielded mutant animals at a high frequency and the unc-22 IV gene is a preferred mutational target. The forward spontaneous mutation frequency at the unc-22 locus in Bergerac BO is about 1 x 10^-4 , and most of these spontaneous unc-22 mutations revert at frequencies between 2 x 10^-3 and 2 x 10 ^-4. Both the forward mutation frequency and the reversion frequency are sensitive to genetic background. Spontaneous unc-22 mutations derived in a Bergerac background and placed in a primarily Bristol background revert at frequencies of <10^-6. When reintroduced into a Bergerac/Bristol hybrid background the mutations once again become unstable.

The mutator activity could not be localized to a discrete site in the Bergerac genome. Nor did mutator activity require the Bergerac unc-22 gene as a target since the Bristol unc-22 homolog placed in a Bergerac background also showed high mutation frequency. Intragenic mapping of two spontaneous unc-22 alleles, st136 and st137, place both mutations in the central region of the known unc-22 map. However, these mutations probably recombine with one another, suggesting that the unstable mutations can occur in more than one site in unc-22. Examination of the phenotypic effect of these mutations on muscle structure indicates that they are less severe in their effect than a known amber allele. We suggest that this mutator system is polygenic and dispersed over the nematode genome and could represent activity of the transposable element Tc1.

     

Group A streptococcal phage T12 carries the structural gene for pyrogenic exotoxin type A

Summary In this paper we describe the meiotic pairing behavior of two free duplications in Caenorhabditis elegans. sDp1 is a duplication of approximately 30 map units of the right portion of linkage group I including unc-74 to unc-54. This duplication pairs, recombines, and apparently segregates from one of the normal homologues. A second duplication, sDp2, is a duplication of approximately 15 map units of the left portion of the linkage group. sDp2 was not observed to recombine with the normal homologue but did suppress exchange between the two normal homologues in a sDp2/ ++ / dpy-5 unc-35 heterozygote. Although a number of free duplications have been described previously in Caenorhabditis elegans, none of these have been shown to pair with normal homologues. The meiotic behavior of the duplications described in this paper can be understood assuming the existence in C. elegans chromosomes of pairing sites of the type described in D. melanogaster chromosomes (I. Sandler 1956; Hawley 1980).     

Retrofitting ampicillin resistant vectors by recombination for use in generating C. elegans transgenic animals by bombardment

Caenorhabditis elegans is an important model organism for modern biologic research. An essential aspect of C. elegans research is the production of transgenic animals for study. These are often generated via microinjection, but biolistic bombardment has become increasingly popular. However, many of the plasmids previously generated for use in microinjection are not readily used for bombardment due to the lack of a convenient marker. The unc-119 gene is often used as a marker since unc-119 rescue can be observed at low magnification, allowing rescued animals to be easily distinguished from the larger number of non-rescued animals. Here we report the use of homologous recombination in Escherichia coli as a method to insert a cassette containing the unc-119 gene into commonly used plasmids at the site of the ampicillin resistance gene which is simpler than other methods like subcloning. These cassettes are flanked by regions homologous to the 5′ and 3′ ends of the ampicillin resistance gene and contain either the unc-119 gene and the kanamycin resistance gene or a unc-119:mCherry fusion gene and the kanamycin resistance gene. The resulting plasmids may be used for biolistic bombardment to yield animals that display unc-119 rescue, and also express the recipient plasmid transgene.     

Genetic and fine structure analysis of unc-26(IV) and adjacent regions in Caenorhabditis elegans

Summary The genetic organization of unc-26(IV) and adjacent regions was studied in Caenorhabditis elegans. We constructed a fine structure genetic map of unc-26(IV), a gene that affects locomotion and pharyngeal muscle movement but not muscle structure. Eleven alleles were positioned relative to each other recombinationally and were classified according to phenotypic severity. The unc-26 gene spans at least 0.026 map units, which is exceptionally large for a C. elegans gene. All but one allele, e205, are amorphic alleles. Interestingly, e205 is hypomorphic but also suppressible by the amber suppressor sup-7. Nineteen lethal mutations in the unc-26 region were isolated and characterized. The unc-26 region is subdivided into four zones by five deficiency breakpoints. These mutations fall into 15 complementation groups. The stages of development affected by these mutations were determined.     

Molecular Characterization of a Soybean Cyst Nematode (Heterodera glycines) Homolog of unc-87

In Caenorhabditis elegans the unc-87 gene encodes a protein that binds to actin at the I band and is important in nematodes for maintenance of the body-wall muscle. Caenorhabditis elegans mutant phenotypes of unc-87 exhibit severe paralysis in larvae and limp paralysis in the adult. We cloned and characterized a full-length cDNA representing a Heterodera glycines homolog of the unc-87 gene from C. elegans that encodes a protein that contains a region of seven repeats similar to CLIK-23 from C-elegans and has 81% amino acid identity with that of C. elegans unc-87 variant A. In the EST database clones labeled "unc-87'''' encode mainly the 3'' portion of unc-87, while clones labeled "calponin homolog OV9M'''' contain mainly DNA sequence representing the 5'' and middle transcribed regions of unc-87. A 1770 nucleotide cDNA encoding H. glycines unc-87 was cloned and encodes a predicted UNC-87 protein product of 375 amino acids. The expression of unc-87 was determined using RT-PCR and, in comparison to its expression in eggs, unc-87 was expressed 6-fold higher in J2 juveniles and 20-fold and 13-fold (P = 0.05) higher in nematodes 15 and 30 days after inoculation, respectively. In situ hybridization patterns confirmed the expression patterns observed with RT-PCR.     

The Unc-22(iv) Region of Caenorhabditis Elegans: Genetic Analysis of Lethal Mutations

The organization of essential genes in the unc-22 region, defined by the deficiency sDf2 on linkage group IV, has been studied. Using the balancer nT1 (IV;V), which suppresses recombination over 49 map units, 294 lethal mutations on LGIV(right) and LGV(left) were recovered using EMS mutagenesis. Twenty-six of these mutations fell into the unc-22 region. Together with previously isolated lethal mutations, there is now a total of 63 lethal mutations which fall into 31 complementation groups. Mutations were positioned on the map using eight overlapping deficiencies in addition to sDf2. The lethal alleles and deficiencies in the unc-22 region were characterized with respect to their terminal phenotypes. Mapping of these lethal mutations shows that sDf2 deletes a minimum of 1.8 map units and a maximum of 2.5 map units. A minimum estimate of essential gene number for the region using a truncated Poisson calculation is 48. The data indicate a minimum estimate of approximately 3500 essential genes in the Caenorhabditis elegans genome.     

Molecular cloning and characterization of the dpy-20 gene of Caenorhabditis elegans

We describe the molecular analysis of the dpy20 gene in Caenorhabditis elegans. Isolation of genomic sequences was facilitated by the availability of a mutation that resulted from insertion of a Tc1 transposable element into the dpy-20 gene. The Tc1 insertion site in the m474:: Tc1 allele was identified and was found to lie within the coding region of dpy-20. Three revertants (two wild-type and one partial revertant) resulted from the excision of this Tc1 element. Genomic dpy-20 clones were isolated from a library of wild-type DNA and were found to lie just to the left of the unc-22 locus on the physical map, compatible with the position of dpy-20 on the genetic map. Cosmid DNA containing the dpy-20 gene was successfully used to rescue the mutant phenotype of animals homozygous for another dpy-20 allele, e1282ts. Sequence analysis of the putative dpy-20 homologue in Caenorhabditis briggsae was performed to confirm identification of the coding regions of the C. elegans gene and to identify conserved regulatory regions. Sequence analysis of dpy-20 revealed that it was not similar to other genes encoding known cuticle components such as collagen or cuticulin. The dpy-20 gene product, therefore, identifies a previously unknown type of protein that may be directly or indirectly involved in cuticle function. Northern blot analysis showed that dpy-20 is expressed predominantly in the second larval stage and that the mRNA is not at all abundant. Data from temperature shift studies using the temperature-sensitive allele e1282ts showed that the sensitive period also occurs at approximately the second larval stage. Therefore, expression of dpy-20 mRNA and function of the DPY-20 protein are closely linked temporally.     

The major gut esterase locus in the nematode Caenorhabditis elegans

Summary Mutations in the major gut esterase of the nematode Caenorhabditis elegans have been induced by ethylmethane sulfonate and detected by isoelectric focusing. The gut esterase locus, denoted ges-1, maps less than 0.3 map units to the right of the unc-60 locus, at the left end of chromosome V.     

Choline Acetyltransferase-Deficient Mutants of the Nematode CAENORHABDITIS ELEGANS

We have identified five independent allelic mutations, defining the gene cha-1, that result in decreased choline acetyltransferase (ChAT) activity in Caenorhabditis elegans. Four of the mutant alleles, when homozygous, lead to ChAT reductions of>98%, as well as recessive phenotypes of uncoordinated behavior, small size, slow growth and resistance to cholinesterase inhibitors. Animals homozygous for the fifth allele retain approximately 10% of the wild-type enzyme level; purified enzyme from this mutant has altered K_m values for both choline and acetyl-CoA and is more thermolabile than the wild-type enzyme. These qualitative alterations, together with gene dosage data, argue that cha-1 is the structural gene for ChAT. cha-1 has been mapped to the left arm of linkage group IV and is within 0.02 map unit of the gene unc-17, mutant alleles of which lead to all of the phenotypes of cha-1 mutants except for the ChAT deficiency. Extensive complementation studies of cha-1 and unc-17 alleles reveal a complex complementation pattern, suggesting that both loci may be part of a single complex gene.     

Copulation in C. elegans males requires a nuclear hormone receptor

In Caenorhabditis elegans, uncoordinated (unc)-55 encodes a nuclear hormone receptor that is necessary for coordinated movement and male mating. An unc-55 reporter gene revealed a sexually dimorphic pattern: early in post-embryonic motor neurons in both sexes; and later in a subset of male-specific cells that included an interneuron and eight muscle cells. A behavioral analysis coupled with RNA interference (RNAi) revealed that males require UNC-55 to execute copulatory motor programs. Two mRNA isoforms (unc-55a and unc-55b) were detected throughout post-embryonic development in males, whereas only one, unc-55a, was detected in hermaphrodites. In unc-55 mutant males isoform a rescued the locomotion and mating defect, whereas isoform b rescued the mating defect only. Isoform b represents the first report of male-specific splicing in C. elegans. In addition, isoform b extended the number of days that transgenic unc-55 mutant males mated when compared to males rescued with isoform a, suggesting an anabolic role for the nuclear hormone receptor. The male-specific expression and splicing is part of a regulatory hierarchy that includes two key genes, male abnormal (mab)-5 and mab-9, required for the generation and differentiation of male-specific cells. We suggest that UNC-55 acts as an interface between genes involved in male tail pattern formation and those responsible for function.     

unc-93(e1500): A Behavioral Mutant of CAENORHABDITIS ELEGANS That Defines a Gene with a Wild-Type Null Phenotype

The uncoordinated, egg-laying-defective mutation, unc-93(e1500) III, of the nematode Caenorhabditis elegans spontaneously reverts to a wild-type phenotype. We describe 102 spontaneous and mutagen-induced revertants that define three loci, two extragenic (sup-9 II and sup-10 X) and one intragenic. Genetic analysis suggests that e1500 is a rare visible allele that generates a toxic product and that intragenic reversion, resulting from the generation of null alleles of the unc-93 gene, eliminates the toxic product. We propose that the genetic properties of the unc-93 locus, including the spontaneous reversion of the e1500 mutation, indicate that unc-93 may be a member of a multigene family. The extragenic suppressors also appear to arise as the result of elimination of gene activity; these genes may encode regulatory functions or products that interact with the unc-93 gene product. Genes such as unc-93, sup-9 and sup-10 may be useful for genetic manipulations, including the generation of deficiencies and mutagen testing.     

Homologous gene targeting in Caenorhabditis elegans by biolistic transformation

Targeted homologous recombination is a powerful approach for genome manipulation that is widely used for gene alteration and knockouts in mouse and yeast. In Caenorhabditis elegans, several methods of target-selected mutagenesis have been implemented but none of them provides the opportunity of introducing exact predefined changes into the genome. Although anecdotal cases of homologous gene targeting in C.elegans have been reported, no practical technique of gene targeting has been developed so far. In this work we demonstrate that transformation of C.elegans by microparticle bombardment (biolistic transformation) can result in homologous recombination between introduced DNA and the chromosomal locus. We describe a scaled up version of biolistic transformation that can be used as a method for homologous gene targeting in the worm.     

Mutants with altered muscle structure in Caenorhabditis elegans

In the small nematode, Caenorhabditis elegans, mutants with a disorganized myofilament lattice structure have been identified by polarized light and electron microscopy. Genetic analysis places the mutations in 12 complementation groups which are distributed over the six linkage groups of C. elegans. The phenotypes are described for the mutants from the 9 complementation groups not previously reported on in detail. Most are paralyzed, but some exhibit essentially normal movement; mutants of two loci show changes only in later larval stages and adulthood. Morphological studies show that, in general, all the members of a complementation group show similar changes in muscle structure and that these changes are distinctive for that group. In mutants of several genes, disorganization of the myofilament lattice is general with no one component of the lattice more obviously altered than others. In mutants of other genes specific structures are prominently altered. In one of the instances where thick filaments appear to be abnormal, double mutants combining mutations in this gene (unc-82 IV) with mutations in the gene for a myosin heavy chain (MacLeod et al., 1977a,b) or paramyosin (Waterston et al., 1977) were used to show that the unc-82 gene product probably affects thick filament assembly through its actions on paramyosin. Some possible implications of the morphological features of the mutants as well as the conclusions derived from the genetic studies are discussed.     

Molecular analysis of two genes between let-653 and let-56 in the unc 22(IV) region of Caenorhabditis elegans

Summary A previous study of genomic organization described the identification of nine potential coding regions in 150 kb of genomic DNA from the unc-22(IV) region of Caenorhabditis elegans. In this study, we focus on the genomic organization of a small interval of 0.1 map unit bordered on the right by unc-22 and on the left by the left-hand breakpoints of the deficiencies sDf9, sDf19 and sDf65. This small interval at present contains a single mutagenically defined locus, the essential gene let-56. The cosmid C11F2 has previously been used to rescue let-56. Therefore, at least some of C11F2 must reside in the interval. In this paper, we report the characterization of two coding elements that reside on C11F2. Analysis of nucleotide sequence data obtained from cDNAs and cosmid subclones revealed that one of the coding elements closely resembles aromatic amino acid decarboxylases from several species. The other of these coding elements was found to closely resemble a human growth factor activatable Na⁺/H⁺ antiporter. Pairs of oligonucleotide primers, predicted from both coding elements, have been used in PCR experiments to position these coding elements between the left breakpoint of sDf19 and the left breakpoint of sDf65, between the essential genes let-653 and let-56.     

The DH-PH region of the giant protein UNC-89 activates RHO-1 GTPase in Caenorhabditis elegans body wall muscle

Mutation of the Caenorhabditis elegans gene unc-89 results in disorganization of muscle A-bands. unc-89 encodes a giant polypeptide (900 kDa) containing a DH domain followed by a PH domain at its N terminus, which is characteristic of guanine nucleotide exchange factor proteins for Rho GTPases. To obtain evidence that the DH-PH region has activity toward specific Rho family small GTPases, we conducted an experiment using the yeast three-hybrid system. The DH-PH region of UNC-89 has exchange activity for RHO-1 (C. elegans RhoA), but not for CED-10 (C. elegans Rac), MIG-2 (C. elegans RhoG), or CDC-42 (C. elegans Cdc42). The DH domain alone has similar activity for RHO-1. An in vitro binding assay demonstrates interaction between the DH-PH region of UNC-89 and each of the C. elegans Rho GTPases. Partial knockdown of rho-1 in C. elegans adults showed a pattern of disorganization of myosin thick filaments similar to the phenotype caused by unc-89 (su75), a mutant allele in which all of the isoforms containing the DH-PH region are missing. Taken together, we propose a model in which the DH-PH region of UNC-89 activates RHO-1 GTPase for organization of myosin filaments in C. elegans muscle cells.