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Mutations affecting embryonic cell migrations in Caenorhabditis elegans.

Four recessive mutations that affect long-range embryonic migration of the two canal-associated neurons (CANs) in C. elegans were isolated and characterized with the goal of identifying genes involved in control of directed cell movement. Mutant animals were identified initially by their "withered" tails, a phenotype associated with abnormal CAN migration; the mutants were then analyzed for abnormal cell migrations by Nomarski microscopy. Based on genetic complementation tests, the mutations were assigned to four different loci, two new (mig-10 III, mig-11 III) and two previously identified (unc-39 V, vab-8 V). Mutations at all four loci affect CAN migration with high to moderate penetrance (the percentage of mutant animals that exhibit the phenotype). In addition, two other bilaterally symmetric pairs of neurons (ALM and HSN), the mesoblast M, and a pair of coelomocyte mother cells are affected by one or more of the mutations, generally with lower penetrance. With the exceptions of HSN and the right coelomocyte mother cell, which occasionally migrate beyond their normal destinations, the cells affected appear to migrate either incompletely or not at all. All the migration phenotypes show incomplete penetrance and variable expressively, although genetic tests suggest that mutations at mig-10 and vab-8 result in complete or nearly complete loss of gene function. The variability in mutant phenotypes allowed tests for interdependence of several of the affected migrations; all those analyzed appeared independent of one another. The possible nature of the mutant defects and possible roles of these four loci in cell migration are discussed.

Animals↗

Genetic complementation in apicomplexan parasites.

A robust forward genetic model for Apicomplexa could greatly enhance functional analysis of genes in these important protozoan pathogens. We have developed and successfully tested a genetic complementation strategy based on genomic insertion in Toxoplasma gondii. Adapting recombination cloning to genomic DNA, we show that complementing sequences can be shuttled between parasite genome and bacterial plasmid, providing an efficient tool for the recovery and functional assessment of candidate genes. We show complementation, gene cloning, and biological verification with a mutant parasite lacking hypoxanthine-xanthine-guanine phosphoribosyltransferase and a T. gondii cDNA library. We also explored the utility of this approach to clone genes based on function from other apicomplexan parasites using Toxoplasma as a surrogate. A heterologous library containing Cryptosporidium parvum genomic DNA was generated, and we identified a C. parvum gene coding for inosine 5-monophosphate-dehydrogenase (IMPDH). Interestingly, phylogenetic analysis demonstrates a clear eubacterial origin of this gene and strongly suggests its lateral transfer from epsilon-proteobacteria. The prokaryotic origin of this enzyme might make it a promising target for therapeutics directed against Cryptosporidium.

Animals↗

A new beige mutant rat ACI/N-Lystbg-Kyo.

A new beige-like coat color mutant was identified in the ACI/N rat colony. Other features characteristic of beige mutants, such as giant granule cells in various tissues, and prolonged bleeding time were also observed. The genetic complementation test, mating beige-like mutant with the authentic beige mutant rat, DA/Ham-Lystbg, revealed that the mutant gene is allelic to Lystbg. The new beige mutant allele was denoted Lystbg-Kyo. Molecular genetic analysis revealed deletion of exons 28, 29, and 30 of the Lyst gene owing to recombination between L1 elements in the mutant rats. Although the deletion was similar to that identified in DA/Ham-Lystbg rats, the putative deletion break points in L1 elements were different in the two strains. Further characterization of the ACI/N-Lystbg-Kyo rats should make it useful as an animal model for human Chediak-Higashi syndrome.

Alleles↗

Cloning of sporulation gene spoOB of Bacillus subtilis and its genetic and biochemical analysis.

A specialized transducing phage carrying a sporulation gene (spoOB) was constructed from Bacillus subtilis temperate phage rho 11 by in vitro and in vivo recombinations. Transformation experiments showed that the spoOB gene resides on a 1.4-megadalton fragment generated by EcoRI endonuclease treatment of the phage deoxyribonucleic acid (DNA). Mutants of this phage which lost transducing activity were isolated and used for genetic complementation tests and the analysis of protein(s) coded by the 1.4-megadalton fragment. The spoOB locus was shown to be composed of one cistron. Sodium dodecyl sulfate-polyacrylamide gel analysis of proteins synthesized in ultraviolet-irradiated cells infected with these phages showed that the 1.4-megadalton fragment codes at least one protein, of molecular weight 39,000, which is synthesized in both vegetative and sporulating cells. A cleavage map of the phage DNA was constructed by use of restriction endonucleases, EcoRI, BamHI, and SalI, and the site of integration of the 1.4-megadalton fragment was determined. Expression and function of the spoOB gene are discussed.

Bacillus subtilis↗

Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus.

Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia. The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome. Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus. The sym8 mutants are arrested during infection by the bacteria early in the developmental process. The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed. These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L. japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti. Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles. In the screen reported here independent symbiotic loci thus appeared with a frequency of approximately 1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction.

Crosses, Genetic↗

Analysis of Tn7 transposition.

Five gene products required for Tn7 transposition were identified using genetic complementation tests. Four of these (tnsA, tnsB, tnsC and tnsD) are essential for insertion into the attachment site, whereas the fifth (tnsE) is required for transposition to plasmids which lack this site. tnsD is not required for transposition to plasmids lacking the attachment site. This analysis used a chloramphenicol-resistant 'mini' transposon containing Tn7 termini but no complete Tn7 gene product and several compatible expression vectors containing cloned Tn7 fragments. The use of transcriptional and translational fusions allowed the identification of two promoters (P1 and P2) at the righthand end of the transposon and indicated that at least tnsA, tnsB and tnsC are translated in the same direction. Expression from P1 appears to be repressed by tnsB.

Cloning, Molecular↗

An interspecific functional complementation test in Drosophila for introductory genetics laboratory courses.

Introductory genetics courses often include evolutionary genetics concepts such as sequence homology and functional conservation. It is usually assumed that two sequences showing homology (i.e., sharing a common ancestral sequence) perform the same molecular function. The correlation, however, does not always hold true, and evidence for functional conservation must come from functional studies. In this study we describe a genetics laboratory class that demonstrates functional conservation between the Drosophila protein Muscleblind (Mbl) and its human ortholog MBNL1. We use the Gal4/UAS system to express MBNL1 in a Drosophila mutant background and measure the in vivo activity of the human protein by rescue of mbl mutant phenotype in embryos. As a control, ubiquitous expression of Drosophila MblC, one of the four protein isoforms encoded by the gene, increased by 71% the viability of mbl mutant embryos and greatly reduced the hypercontracted abdomen of mutant larvae. In a parallel experiment, human MBNL1 provided a robust rescue of the embryonic lethality (78%) and improved abdomen hypercontraction as well. Under both conditions, rescued larvae die as first instars, probably due to overexpression effects, lack of alternative protein isoforms, or incomplete expression in critical tissues such as the nervous system. The use of two constructs in the rescue experiment (UAS-mblC and UAS-MBNL1) and the incomplete rescue prompt several questions for students. The fact that a human protein works in a Drosophila cellular context illustrates the use of an in vivo test to prove functional conservation.

Amino Acid Sequence↗

The araC regulatory gene mRNA contains a leader sequence.

An estimation of the size of the araC gene in Escherichila coli B/r was made by sub-cloning restriction fragments of the araC-containing hybrid plasmid pTB1 into the plasmid pBR322. Plasmids which contained a functional araC gene were identified by genetic complementation tests. DNA sequence analysis of the promoter-proximal region of the araC gene revealed that araC mRNA contains a 150 nucleotide leader.

Arabinose↗

napts, a mutation affecting sodium channel activity in Drosophila, is an allele of mle, a regulator of X chromosome transcription.

napts is a recessive mutation that affects the level of sodium channel activity and, at high temperature, causes paralysis associated with a loss of action potentials. We show, by genetic complementation tests, germline transformation, and analysis of mutations, that napts is a gain-of-function mutation of mle, a gene required for X chromosome dosage compensation and male viability. Molecular analyses of nap and mle mutations indicate that mle+, nap+, and napts activities are encoded by the same open reading frame and suggest that napts is due to a single amino acid substitution. Although napts is known to act via para+, an X-linked sodium channel structural gene, its effect is not due to a simple defect in para+ dosage compensation.

Action Potentials↗

Transcriptional analysis of type 1 capsule genes in Staphylococcus aureus.

Serotype 1 capsule of Staphylococcus aureus plays an important role in staphylococcal infections. A 14.6 kb chromosomal DNA fragment containing 13 cap1 genes responsible for the synthesis of type 1 capsule in S. aureus strain M has been previously cloned and sequenced in our laboratory. These genes are closely linked and are transcribed in one orientation. To study the transcription of these genes, we employed genetic complementation using a set of plasmids with various deletions in the 14.6 kb region to complement mutants mapped in each of the 13 genes. We found that there were six transcriptional units. By Northern hybridization using the entire gene cluster as a probe, we identified a large 14 kb band and several smaller bands ranging from 0.3 to 6 kb. The 14 kb band was also identified by various individual gene probes, suggesting that all 13 genes are transcribed into a large transcript. The 14 kb transcript and the smaller bands were not detected when a 533 bp fragment containing the potential promoter region upstream of the first gene (cap1A) was deleted from the chromosome, suggesting that the small transcripts are the degradation (processing) products of the 14 kb transcript. The activities of the promoters identified by genetic complementation tests were quantitatively measured by transcriptional fusions using xylE as the reporter gene. The result showed that the promoter preceding the first cap1 gene was 45-198-fold higher than the downstream promoters. The result from gene-fusion studies is in agreement with the Northern analysis in that only the transcript transcribed from the first promoter was detected. Taken together, these results suggest that the cap1 genes are organized as a large operon with at least five weak internal promoters, and the long polycistronic transcript is processed (degraded) into several smaller transcripts. The transcription initiation site of the primary transcript was mapped by S1 nuclease mapping and the major promoter was defined by deletion analysis of the promoter-xylE fusion. In addition, we showed that the internal promoters, despite their weak activities, were sufficiently effective at expressing their downstream genes required for producing capsule, compared to the wild-type strain.

Bacterial Capsules↗

A case of xeroderma pigmentosum complementation group F with neurological abnormalities.

We report a 48-year-old Japanese man suffering from xeroderma pigmentosum associated with mental retardation, cerebral atrophy and cerebellar ataxia. Cultured fibroblasts from an unexposed area of skin had reduced DNA repair capacity after UV irradiation, with higher sensitivity to UV than normal cells in colony-forming ability and host cell reactivation using herpes simplex virus. Genetic complementation tests by cell fusion with polyethylene glycol revealed that the patient belonged to group F. He died of bile duct cancer at the age of 50. This is the first report of an XP-F patient with neurological abnormalities.

Brain Diseases↗

Genetic interaction between hoxb-5 and hoxb-6 is revealed by nonallelic noncomplementation.

hoxb-5 and hoxb-6 are adjacent genes in the mouse HoxB locus and are members of the homeotic transcription factor complex that governs establishment of the mammalian body plan. To determine the roles of these genes during development, we generated mice with a targeted disruption in each gene. Three phenotypes affecting brachiocervicothoracic structures were found in the mutant mice. First, hoxb-5- homozygotes have a rostral shift of the shoulder girdle, analogous to what is seen in the human Sprengel anomaly. This suggests a role for hoxb-5 in specifying the position of limbs along the anteroposterior axis of the vertebrate body. Second, hoxb-6- homozygotes frequently have a missing first rib and a bifid second rib. The third phenotype, an anteriorizing homeotic transformation of the cervicothoracic vertebrae from C6 through T1, is common to both hoxb-5- and hoxb-6- homozygotes. Quite unexpectedly, hoxb-5, hoxb-6 transheterozygotes (hoxb-5-hoxb-6+/hoxb-5+ hoxb-6-) also show the third phenotype. By this classical genetic complementation test, these two mutations appear as alleles of the same gene. This phenomenon is termed nonallelic noncomplementation and suggests that these two genes function together to specify this region of the mammalian vertebral column.

Alleles↗

A fifth gene (uncE) in the operon concerned with oxidative phosphorylation in Escherichia coli.

Three mutant unc alleles (unc-408, unc-410, and unc-429) affecting the coupling of electron transport to oxidative phosphorylation in Escherichia coli K-12 have been characterized. Genetic complementation analyses using previously defined mutant unc alleles indicated that the new mutant unc alleles affect a previously undescribed gene designated uncE. The phenotype of strains carrying the uncE408 or uncE429 allele is similar in that Mg(2+)-adenosine triphosphatase activity is only found in the cytoplasmic fraction, and membranes do not bind the F(1) portion of adenosine triphosphatase purified from a normal strain. In contrast, adenosine triphosphatase activity is present both in the cytoplasm and on the membranes from a strain carrying the unc-410 allele, and normal F(1) binds to F(1)-depleted membranes from this strain. The adenosine triphosphatase solubilized from membranes of a strain carrying the unc-410 allele reconstituted ATP-dependent membrane energization in F(1)-depleted membranes from a normal strain. Genetic complementation tests using various Mu-induced unc alleles in partial diploid strains show that the uncE gene is in the unc operon and that the order of genes is uncB E A D C. The unc-410 allele differs from the uncE408 and uncE429 alleles in that complementation tests with the Mu-induced unc alleles indicate that more than one gene is affected. It is concluded that this is due to a deletion which includes part of the uncE gene and another gene, or genes, between the uncE and uncA genes.

Adenosine Triphosphatases↗

Epistatic suppression of systemic lupus erythematosus: fine mapping of Sles1 to less than 1 mb.

Sle is a susceptibility locus for systemic autoimmunity derived from the lupus-prone NZM2410 mouse. The New Zealand White-derived suppressive modifier Sles1 was identified as a specific modifier of Sle1 and prevents the development of IgG anti-chromatin autoantibodies mediated by Sle1 on the C57BL/6 (B6) background. Fine mapping of Sles1 with truncated congenic intervals localizes it to a approximately 956-kb segment of mouse chromosome 17. Sles1 completely abrogates the development of activated T and B cell populations in B6.Sle1. Despite this suppression of the Sle1-mediated cell surface activation phenotypes, B6.Sle1 Sles1 splenic B cells still exhibit intrinsic ERK phosphorylation. Classic genetic complementation tests using the nonautoimmmune 129/SvJ mouse suggests that this strain possesses a Sles1 allele complementary to that of New Zealand White, as evidenced by the lack of glomerulonephritis, splenomegaly, and antinuclear autoantibody production seen in (129 x B6.Sle1 Sles1)F(1)s. These findings localize and characterize the suppressive properties of Sles1 and implicate 129 as a useful strain for aiding in the identification of this elusive epistatic modifier gene.

Animals↗

The organization of the purL gene encoding 5'-phosphoribosylformylglycinamide amidotransferase of Escherichia coli.

Escherichia coli 5'-phosphoribosylformylglycinamide (FGAR) amidotransferase (EC 6.3.5.3) encoded by the purL gene catalyzes the conversion of FGAR to formylglycinamidine in the presence of glutamine and ATP for the de novo purine nucleotide biosynthesis. On the basis of the nucleotide sequence of purL, the enzyme was dissected along the polypeptide chain into at least three discrete regions, designated as domains I, II, and III, by genetic complementation tests. Domain III (255 amino acids), which resides in the C-terminal region, is similar in amido acid sequence to several glutamine amidotransferases and exerts the transfer of the amide nitrogen of glutamine. Domain I (791 amino acids) resides in the N-terminal region and contains a potential ATP binding motif. Domain II (249 amino acids) locates between domains I and III and is composed of an alternating structure of at least eight predicted beta-strand and alpha-helix elements, as has been observed in the family of triosephosphate isomerases. The functions of domains I and II have been discussed in relation to the transfer of the carbonyl oxygen of FGAR into the gamma-phosphorus moiety of ATP. These results support a model that the E. coli purL gene is a fused gene of at least three different gene families. The highly repetitive sequences of the E. coli genome appeared to play an important role in the process of the gene fusion.

Amino Acid Sequence↗

Transposon 10 promoted deletions and inversions in the transfer genes of R100-1.

Spontaneous tetracycline-sensitive, transfer-deficient mutants of R100-1 were selected and analysed by genetic complementation tests and with the restriction endonuclease EcoR1. While some of the Tets Tra- mutants were caused by a single deletion event which removed the Tetr genes and extended into the neighbouring transfer genes, other mutants were the result of the deletion of the Tetr genes within Tn10 which was accompanied by an inversion of adjacent DNA sequences. A clustering of deletion and inversion endpoints occurred in the traA gene. Some of the transfer genes of R100-1 were assigned to EcoR1 fragments.

Chromosome Deletion↗

Low diversity of t haplotypes in the eastern form of the house mouse, Mus musculus L.

In previous studies, 13 different recessive embryonic lethal genes have been associated with t haplotypes in the wild mice of the species Mus domesticus. In this communication we have analyzed five populations of Mus musculus for the presence and identity of t haplotypes. The populations occupy geographically distant regions in the Soviet Union: Altai Mountains, western and eastern Siberia, Azerbaijan and Turkmenistan. No t haplotypes were found in mice from eastern Siberia. In the remaining four populations, t haplotypes occurred with frequencies ranging from 0.07 to 0.21. All the t haplotypes extracted from these populations and analyzed by the genetic complementation test were shown to carry the same lethal gene tcl-w73. In one population (that of western Siberia), another lethal gene (tcl-w5) was found to be present on the same chromosome as tcl-w73. This situation is in striking contrast to that found in the populations of the western form of the house mouse, M. domesticus. In the latter species, tcl-w73 has not been found at all and the different populations are characterized by the presence of several different lethal genes. The low diversity of t haplotypes in M. musculus is consistent with lower genetic variability of other traits and indicates a different origin and speciation mode compared to M. domesticus. Serological typing for H-2 antigenic determinants suggests that most, if not all, of the newly described t haplotypes might have arisen by recombination of tw73 from M. musculus with t haplotypes from M. domesticus either in the hybrid zone between the two species or in regions where the two species mixed accidentally.

Animals↗

Complementation between uncF alleles affecting assembly of the F1F0-ATPase complex of Escherichia coli.

A mutant affected in the b subunit (coded by the uncF gene) of the F1F0-ATPase in Escherichia coli was isolated by a localized mutagenesis procedure in which a plasmid carrying the unc genes was mutagenized in vivo. The biochemical properties of cells carrying the uncF515 allele were examined in a strain carrying the allele on a multicopy plasmid and a mutator-induced polar unc mutation on the chromosome. The strain carrying the mutant unc allele was uncoupled with respect to oxidative phosphorylation. Membrane-bound ATPase activity was very low or absent, and membranes were somewhat proton permeable. It was concluded that the F0 sector was assembled. Determination of the DNA sequence of the uncF515 allele showed it differed from wild type in that a G----A substitution occurred at position 392, resulting in glycine being replaced by aspartate at position 131. Genetic complementation tests indicated that the uncF515 allele complemented the uncF476 allele (Gly 9----Asp). Two-dimensional gel electrophoresis of membrane preparations indicated that the uncF515 and uncF476 alleles interrupted assembly of the F1F0-ATPase at different stages.

Adenosine Triphosphatases↗