Genetic instability at the cut locus of Drosophila melanogaster induced by the MR-h12 chromosome

Summary Unstable mutations were generated at the cut locus by the MR-h12 factor which induces male recombination. The unstable allele ct ^MR2, containing the MR-transposon in the cut locus is a very powerful mutator producing a number of different viable and lethal mutations both in the cut locus and outside it.I describe several types of mutations: stable reversion to wild type, which were sometimes associated with the appearance of unstable mutations in other loci; of stable deficiencies at the cut locus (lethals); new unstable mutations at different loci with the ct ^MR2 allele conserved; new unstable cut alleles with a phenotype other than that of ct ^MR2. The possible mechanisms of these mutational events are discussed. The genetic system constructed in the present work affords an opportunity for molecular studies of the cut locus and the MR-transposon, as a sequence from the cut locus has recently been cloned (Tchurikov et al. 1981).     

An Increase in the X-Linked Lethal Mutation Rate Associated with an Unstable Locus in DROSOPHILA MELANOGASTER

The behavior of an unstable allele of the singed-bristle locus on the X chromosome was studied in connection with the occurrence of lethal mutations on that same chromosome. The unstable allele, weak singed (sn^w), is under the control of the P-M system of hybrid dysgenesis and, in the M cytotype, mutates secondarily to extreme singed (sn^e) and to wild type (sn⁺) at high rates. Chromosomes whose sn^w allele had mutated in this fashion sustained lethal mutations at a rate of 3%; whereas, those whose sn^w allele had apparently remained unchanged, acquired lethals at a lower rate, 1.3%. The significant difference between these values indicates a statistical coincidence between the phenomena of sn^w instability and X-linked lethal mutation induction. This coincidence can be explained by postulating that mutations at the singed locus sometimes release a genetic element capable of reinserting elsewhere in the chromosome. Alternately, sn^w instability and lethal induction might be associated because they are the effects of a common cause, perhaps some mutation-inducing substance present in various amounts in the germ cells of dysgenic flies.—The lethals that occurred on chromosomes whose sn^w allele had mutated to sn^e mapped preferentially close to singed. The lethals on the sn^w and sn⁺ chromosomes did not show this concentration on the map. Cytological analysis of samples of all three types of lethal chromosomes indicated that, with one exception, there was no detectable breakage at the singed locus itself. The single instance of breakage at singed was not associated with any change in the singed phenotype. Thus, the instability of sn^w apparently does not involve detectable breakage of the singed locus, or if it does, this breakage is not a common event.     

Five genetic loci involved in the synthesis of acidic exopolysaccharides are closely linked in the genome of Rhizobium sp strain NGR234

Summary R-prime plasmids were constructed from a derivative of Rhizobium strain NGR234 (ANU280) and were shown to contain overlapping genomic DNA segments involved in biosynthesis of exopolysaccharides (EPS). The R-primes originally constructed carried the mutant allele from Tn5-induced EPS-deficient (Exo^–) mutant ANU2811. This plasmid-located mutant allele was dominant to the corresponding wild-type allele as merodiploid strains were Exo^–. Exo⁺ revertants occurred at a low rate (1×10^-7) and these were shown to result from double reciprocal recombination events, which led to the isolation of R-prime plasmids carrying functional wild-type exo alleles. R-prime plasmids that carry overlapping segments of DNA from parental strain ANU280 complemented 28 of the 30 group 2 Exo^– mutants of strain ANU280. Complementation of these Exo^– mutants also restored their symbiotic abilities of effective nodulation. Subsequent in vivo recombination between the wild-type alleles located on the R-prime and the corresponding mutated allele on the genome, was used to generate a new family of R-primes, which carried mutations in the exo genes. The 30 group 2 Exo^– mutants were classified into 7 distinct genetic groups based upon complementation and physical mapping data. Five of the seven exo loci were gentically linked and located on a 15-kb region of DNA. Mutations at two loci were dominant only when the mutations were R-prime plasmid-located while a mutation at a second locus was cis-dominant to two other exo loci. At least five genes involved in the synthesis of acidic exopolysaccharide synthesis have been identified.     

Reactivation of the ATCase domain of the URA2 gene complex: a positive selection method for Ty insertions and chromosomal rearrangements in Saccharomyces cerevisiae

Genetic rearrangements such as deletions or duplications of DNA sequences are rarely detected in the yeast Saccharomyces cerevisiae. We have developed a screening system using the URA2 gene coding for the bi-functional CPSase-ATCase (carbamyl phosphate synthetase — aspartate transcarbamylase) to select positively for these kinds of events. Nonsense mutations in the CPSase region cause a complete loss of the ATCase activity because of their strong polar effect. Thirty-seven ATCase⁺ revertants were isolated from a strain containing three nonsense mutations in the proximal CPSase region. Genetic and structural analysis of the URA2 locus in these strains allowed us to characterize two major classes of revertants. In the first, an entire copy of a Ty transposon was found to be inserted in the CPSase coding domain. This event, which represents a new form of Ty-mediated gene activation was further analysed by mapping the Ty integration site in 26 strains. In a second class of revertants, we observed chromosomal rearrangements and, in particular, duplication of the ATCase region and its integration in a new chromosomal environment in which this sequence becomes active.     

Construction in Escherichia coli K12 of derivatives of sex factor F143 carrying lexB and recA mutations

Summary Various F sex factors have been derived from F143, an episome extending from lysA to pheA. F143 derivatives carrying recA and lexB alleles and also mutations in genes thyA, argA, cysC were constructed as follows. Recombination was used as a means to generate genetically labelled F-primes. Using trimethoprim as agent of counterselection of Thy⁺ cells in thyA ^–/F-thyA ⁺bacteria, it was possible to collect, after transfer, F-primes modified by deletion of the thyA region or recombination between chromosome and episome. F-primes which had spontaneously recombined with the chromosome and integrated chromosomal markers, were also selected by transfer to proper F^– recipients. P1 transduction of a dominant marker allele into a strain homodiploid for a recessive allele was used to construct F-primes carrying mutations introduced by cotransduction. These F-primes have been useful to establish the dominance and complementation pattern of recA and lexB mutations (Morand, Goze and Devoret, accompanying paper; Glickman, Guijt and Morand, accompanying paper).     

Cytogenetic analysis of chromosome region 89A of Drosophila melanogaster: isolation of deficiencies and mapping of Po, Aldox-1 and transposon insertions

Summary We have initiated a cytogenetic analysis of chromosome region 89A of Drosophila melanogaster by isolating a set of radiation-induced mutations causing loss of function of P[(w)B]1-1, a transposon bearing the white locus inserted in 89A. Complementation tests and cytological examination of these chromosomes identified four new deficiencies (Df(3R)Po ², Df(3R)Po ³, Df(3R)Po ⁴ and Df(3R)c(3)G ² ). The new deficiencies and three previously identified deficiencies (Df(3R)sbd ²⁶, Df(3R)sbd ⁴⁵ and Df(3R)sbd ¹⁰⁵) were tested for the ability to complement mutations in the enzyme loci Po and Aldox-1, the indirect flight muscle genes Tm2 and act88F, the morphological mutations jvl, sbd ² and Sb, the vital loci srp, pnr and mor, and a newly described vital locus l(3)89Aa. We also used linkage analysis to determine the order and relative positions of P[(w)B]1-1 and an independent transposon insertion, P[w⁺]21, with respect to cv-c, Po, Aldox-1 and sbd ². Cytological examination of the deficiencies and analysis of the transformed lines by in situ hybridization permits the correlation of genetically defined regions with specific polytene chromosome bands. A revised cytogenetic map of the 8817–8913 region is presented.     

Spm and I element changes with the a-m2 8004 allele in maize

Summary Among the mobile element systems in maize, the En (Spm) system (En — the regulatory element and I the receptive element — a nonfunctional En) has several interesting aspects of control of gene expression (En and Spm are homologous in structure and activity). One of the alleles arising from the Spm group included the a-m2 8004 allele that has a low spotting pattern and unique ringed areas. The interest in this allele is that Spm or En will induce it to co-express the A phenotype and mutability. Several exceptions of the allele were analyzed. Two are Spm changes and two are I changes. The analysis shows that the heritable changes include I changes that are co-expressed in various grades of color and different degrees of mutability. All these changes occur with I at the locus. The Spm changes also include changes in mutability patterns and a mottling pattern.Journal Paper No., J-11792 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 2381     

Developmental genetics of a temperature-sensitive RNA polymerase II mutation in Drosophila melanogaster

Summary Purified RNA polymerase II (RNA nucleotidyl-transferase; EC 2.7.7.6) extracted from flies possessing lesions in the Ultrabithorax-like (Ubl) locus of Drosophila melanogaster has altered activity in vitro (Greenleaf et al. 1979, 1980; Coulter and Greenleaf 1982). This strongly suggests that the Ubl locus encodes a subunit of RNA polymerase II. Ethyl methanesulfonate was used to induce a temperature-sensitive mutation in this locus. Flies either homozygous or hemizygous for this new X–linked mutation (Ubl ^ts) display viability comparable to that of wild-type flies at 22° C but are lethal at 29° C. The temperature-sensitive period for Ubl ^ts flies is between gastrulation (6 h, 29° C) and pupation (9–10 days, 22° C). Zygotes shifted from 22° C to 29° C die at either the late embryonic or first larval instar stage while temperature shifts of second and third instar larvae result in the lethal phase occurring at the pupal stage. Most pupae shifted from 22° C to 29° C undergo metamorphosis and eclose as adults. Adults are viable if placed at 29° C; however, all females and some males become sterile if maintained at this temperature.Somatic recombination was used to induce clones homozygous for a null allele of Ubl at different stages of development. Clones of this null allele appear to be cell lethal indicating that the Ubl ⁺ gene product is required at all stages of development. The viability of Ubl ^ts pupae and adults at 29° C may result from only a partial reduction in activity caused by the mutation at this nonpermissive temperature.     

Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168

Summary We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB: : cat, leuB ^–, and pro: : neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB ⁺ pro⁺)(leuB: : cat pro ⁺) (leuB ^– pro: : neo)(leuB ^– pro ⁺) resulted in a leuB ^– single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD ^– and pro ^–, as well as the double mutation leuB ^– pro ^– can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.     

Hyper-recombination in dam mutants of Escherichia coli K-12

Summary F-prime heterogenotes of dam-3 bacteria segregate F-prime homogenotes at a frequency 30–200 times higher than the isogenic dam ⁺ strain. A hyperrecombination mutant which shows increased recombination between chromosomal duplications was characterized as a dam mutant. The dam-3 allele causes a reduction in linkage of proximal unselected markers in transconjugants and increases the recombination frequency between a pair of closely linked markers. It is concluded that dam mutations confer a hyperrecombination phenotype to the cell.     

Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): Two gene systems control movement

Summary A large number of motility mutants of the gliding bacterium Myxococcus xanthus have been isolated and analyzed by transduction. Almost all nonmotile mutants are found to be double mutants. This is explained by the existence of two parallel and almost independent multi-gene systems controlling motility, in which case at least one mutation in each system would be required eliminate motility. Only one locus, called mgl, has been found to be shared by both systems. Wild type cells move singly and in groups. Single cells move if they carry a complete gene system A, the genes of which are described in the preceding paper. Groups of cells can move if they carry a complete gene system S, but single A^–S⁺ cells do not move. Linkage analysis reveals at least 9 different loci in system S. One class of S^– mutants, those mutated in the locus tgl, are conditional mutants which, after contact with tgl ⁺ cells, become temporarily motile as cell groups. Most system A mutations have little effect on fruiting but many system S mutations block it, suggesting that system S plays a role in the fruiting process.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Mutations affecting regulation of the anabolic argF and the catabolic aru genes in Pseudomonas aeruginosa PAO

Summary The nucleotide sequence required for a fully functional promoter and operator of the Pseudomonas aeruginosa argF gene (argF _po), the arginine-repressible gene for anabolic ornithine carbamoyltransferase, was defined within a 160 by region. The streptomycin (Sm) resistance genes strAB of plasmid RSF1010 were fused to argF _po. This construct in P. aeruginosa strain PAO conferred resistance to Sm. Mutants of strain PAO were selected which were resistant to Sm in the presence of arginine due to constitutive expression of argF _po —strAB. These mutants were designated argR. They were unable to grow or grew poorly on arginine or ornithine as the sole carbon and nitrogen source. This growth defect (Aru^–/Oru^– phenotype) was correlated with a reduced level of N-succinylornithine aminotransferase, an enzyme participating in the major aerobic pathway for arginine and ornithine catabolism in this organism. The argR mutants were classified into four groups by transduction analysis and three argR mutations were mapped on the PAO chromosome. argR9901 and argR9902 were co-transducible with car-9 (at 1 min) and thus close to the oru-310 locus; argR9906 was localized in the oruI (=aru) gene cluster (67 min). Some aru mutants, which have been isolated previously and which produce very low amounts of all enzymes in the arginine succinyltransferase pathway, were unable to repress the argF gene in an arginine medium. Thus, P. aeruginosa PAO appears to have multiple genes that are involved in the regulation of both the anabolic argF and the catabolic aru genes.Abbreviations Arg^– arginine auxotrophy - Aru arginine utilization - Oru ornithine utilization     

Circadian clock phenotypes of chromosome aberrations with a breakpoint at the per locus

Summary The circadian rhythm phenotypes of eight chromosome aberrations with a breakpoint in the region of the per locus (3B1-2) were analyzed. Two duplications and five deficiencies with a 3B1-2 breakpoint produce either a wild-type or an arrhythmic clock phenotype while one translocation with a 3B1-2 breakpoint, T(1;4)JC43, produces locomotor-activity rhythms with either very-long period (31–39 h), rhythms that grade into arrhythmicity, or completely arrhythmic phenotypes. This is a unique phenotype that had not previously been observed for mutants at the per locus. An extensive complementation analysis of 3B1-2 chromosome aberrations and per mutant alleles provided no compelling evidence for genetic complexity at the per locus. This is in contrast to the report of Young and Judd (1978). Analysis of both the locomotor-activity and eclosion phenotypes of 3B1-2 chromosome aberrations did not uncover differences in the genetic control of these two rhythms. The clock phenotypes of 3B1-2 chromosome aberrations, the three per mutant alleles, and per ⁺ duplications suggest that mutations at the per locus shorten, lengthen, or eliminate periodicity by respectively increasing, decreasing, or eliminating per activity.     

The polymorphism of a novel 30 bp-deletion mutation at KAP9.2 locus in the cashmere goat

The keratins and keratin-associated proteins (KAPs) are a large heterogeneous group of proteins that make up about 90% of the cashmere fiber. Keratin-associated proteins 9.2 gene (KAP9.2) is one of the ultra high sulfur KAPs, which might play an important role in the bundling of intermediate filaments. In this study, the deletion/insertion mutation of KAP9.2 gene in 997 cashmere goat samples was firstly detected, at the same time, parts of these samples were sequenced. The results showed that two alleles were detected at this KAP9.2P1 locus, named allele W and D. The frequencies of the KAP9.2-W allele in Inner Mongolia White cashmere (n = 785) and Shaanbei White cashmere goat breeds (n = 212) were 0.878 and 0.790, respectively. The χ²-test showed that the genotype distributions in these two cashmere goat breeds were not in agreement with Hardy–Weinberg equilibrium. According to the classification of polymorphism information content (PIC), Shaanbei White cashmere goat was more polymorphic at this locus. Moreover a 30 bp-deletion mutation was described at KAP9.2P2 locus for the first time and no deletion/insertion was described at KAP9.2P1 locus. The results possibly revealed that the size polymorphism existed in the two Chinese cashmere goat and the 30 bp-deletion mutation was possibly caused by variations in the number of the decapeptide repeat structures.     

The mouse Clc1/myotonia gene: ETn insertion, a variable AATC repeat, and PCR diagnosis of alleles

Myotonias are muscle diseases in which the function of the muscular chloride channel ClC-1 is impaired. Null alleles of the corresponding Clc1 gene on mouse chromosome (Chr) 6 provide animal models for human myotonias. It was shown that the allele adr (Clc1 ^adr ) is due to an insertion of an ETn type transposon that is transcribed and leads to multiple splicing events; the allele mto (Clc1 ^adr-mto ) involves a stop codon near the N-terminus. We have determined the genomic organization of the mouse Clc1 gene and the sequence requirements for the transposon insertion in the Clc1 ^adr allele. The mouse Clc1 gene is composed of 23 exons, ranging from 39 to 372 bp, and spans approximately 23 kb of genomic DNA. The exon/intron organization is highly homologous to that of the human CLCN1 gene; the homology of the coding sequence is 97% to rat and 89% to human. In the adr allele the ETn transposon is inserted into intron 12, the largest intron. Whereas the 5′ and 3′ LTR sequences of the ETn transposon are homologous to those reported for other insertional mutations of the mouse, no consensus motif for an insertion target site could be defined. On the basis of flanking sequences, we provide duplex PCR diagnoses for the adr, adr-mto, and wild-type alleles of Clc1. Close to the 3′ end of intron 12, a tetranucleotide repeat (AATC)_n was found that is polymorphic between mouse species Mus musculus, M. molossinus, M. castaneus, and M. spretus, and can thus be used for chromosomal mapping studies. Received: 13 October 1996 / Accepted: 10 May 1997     

Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome

To study the variation in spontaneous mutation frequencies in different chromosomal domains, a mini-Mu-kan-lacZ ⁻transposable element was constructed to insert the lacZ ⁻(Trp570 → Opal) allele into many different loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop → LacZ⁺ candidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14 000 colonies, and named plm-1 (preferential lacZmutagenesis) and plm-2. The frequency of LacZ⁻→ LacZ⁺ mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac⁺ reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan^r gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ⁺→ LacZ⁻and Kan^r→ Kan^s mutant frequencies of these Lac⁺ revertants were in the range of 10⁻³ to 10⁻², indicating that this putative localized mutagenesis is neither allele nor gene specific. To identify the plm loci, the chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome. Received: 1 August 1996 / Accepted: 3 April 1997     

The nifU, nifS and nifV gene products are required for activity of all three nitrogenases of Azotobacter vinelandii

Summary Strains with mutations in 23 of the 30 genes and open reading frames in the major nif gene cluster of A. vinelandii were tested for ability to grow on N-free medium with molybdenum (Nif phenotype), with vanadium (Vnf phenotype), or with neither metal present (Anf phenotype). As reported previously, nifE, nifty, nifU, nifS and nifV mutants were Nif^– (failed to grow on molybdenum) while nifM mutants were Nif^–, Vnf^– and Anf^–. nifV, nifS, and nifU mutants were found to be unable to grow on medium with or without vanadium, i.e. were Vnf^– Anf–. Therefore neither vnf nor anf analogoues of nifU, nifS, nifV or nifM are expected to be present in A. vinelandii.     

Analyses oftraA, int-Tn,andxis-TnMutations in the Conjugative Transposon Tn916inEnterococcus faecalis

The conjugative transposon Tn916moves intercellularly via an excision/insertion mechanism that involves products ofint-Tnandxis-Tn.Tn5-insertion mutations in these genes were found to be complemented in anEnterococcus faecalishost by specific coresident transposons harboring the corresponding wild-type allele. A determinant designatedtraA,partially overlapping and divergently transcribed fromxis-Tn,is thought to encode a key positively acting regulatory protein needed for expression of conjugation functions. This locus was also shown to express atrans-acting product.