Regenerant Arabidopsis lineages display a distinct genome-wide spectrum of mutations conferring variant phenotypes

Multicellular organisms can be regenerated from totipotent differentiated somatic cell or nuclear founders [1-3]. Organisms regenerated from clonally related isogenic founders might a priori have been expected to be phenotypically invariant. However, clonal regenerant animals display variant phenotypes caused by defective epigenetic reprogramming of gene expression [2], and clonal regenerant plants exhibit poorly understood heritable phenotypic ("somaclonal") variation [4-7]. Here we show that somaclonal variation in regenerant Arabidopsis lineages is associated with genome-wide elevation in DNA sequence mutation rate. We also show that regenerant mutations comprise?a distinctive molecular spectrum of base substitutions, insertions, and deletions that probably results from decreased DNA repair fidelity. Finally, we show that while regenerant base substitutions are a likely major genetic cause of the somaclonal variation of regenerant Arabidopsis lineages, transposon movement is unlikely to contribute substantially to that variation. We conclude that the phenotypic variation of regenerant plants, unlike that of regenerant animals, is substantially due to DNA sequence mutation.     

Leaky pantothenate and thiamin mutations of Salmonella typhimurium conferring suphometuron methyl sensitivity

The herbicide suphometuron methyl inhibits the utilization of pyruvate and 2-ketobutyrate by the branched-chain amino acid biosynthetic enzyme acetolactate synthase. Eighteen insertions of the transposon Tn10 into the genome of Salmonella typhimurium LT2 caused hypersensitivity to this herbicide. Five of these insertions conferred a partial auxotrophic requirement. Concurrent herbicide sensitivity and heat-labile pantothenate auxotrophy was due to panD::Tn10 mutations, while coincident sulphometuron methyl sensitivity and thiamin auxotrophy was attributable to thiA::Tn10 mutations. The phenotypes of these mutations suggested that coenzyme A and thiamin pyrophosphate availability modulated the cells' response to sulphometuron methyl. A model suggesting a key role for 2-ketobutyrate accumulation in herbicide action is supported by the function of thiamin pyrophosphate in 2-ketoacid metabolism and the known role of a 2-ketoacid in coenzyme A synthesis.     

Two distinct types of mutations conferring to Escherichia coli K12 capability of D-tryptophan utilization

We showed that the ability of Escherichia coli K12 tryptophan auxotrophs to utilize D-tryptophan as a substitute for L-tryptophan may result from two types of mutations. The first type consisted in changes in the dadR regulatory site of the dad operon increasing the synthesis of D-amino acid dehydrogenase. The mutations of the second type mapped within the dad A structural gene. They changed the apparent substrate specificity of D-amino acid dehydrogenase. We suppose that the change may be due to an altered enzyme structure which make it more accessible to D-tryptophan.     

Characterization of a mouse Scube3 reporter line

The SCUBE gene family encode secreted, extracellular proteins that share a distinct domain organization of at least five recognizable motifs, including an amino-terminal signal peptide sequence, multiple EGF-like domains, a large spacer region containing multiple N-linked glycosylation sites, three repeated stretches of six-cysteine residues and a carboxy-terminal CUB domain. We describe a Scube3(tm1Dge/H) targeted allele, which replaces the entire coding region for Exons 2 and 3 with a neomycin-lacZ selectable marker cassette predicted to delete the first two EGF-like domains of the transcribed protein. Scube3(+/tm1Dge/H) embryos demonstrate strong β-galactosidase activity in the early facial epithelium, including the branchial arches and facial processes, the otic vesicle, limb buds, and neural tube. In addition, strong reporter activity was identified in the epithelial compartments of developing teeth and hair follicles. However, analysis of the Scube3(tm1Dge/H) allele revealed that it encodes a truncated protein, which contains part of the spacer region and CUB domain. It is likely that this protein retains functionality because our analysis reveals that Scube3(tm1Dge/H; tm1Dge/H) mice are phenotypically normal. Whilst acting as a useful reporter, these mice do not provide any insight into the potential role of Scube3 during embryonic development.     

Simultaneous detection of multiple mutations conferring streptomycin resistance inMycobacterium tuberculosis using nanoscale engineered biomagnetites

Streptomycin-resistantMycobacterium tuberculosis has been attributed to two distinct classes of mutations, including point mutations within therpsL gene (three mutation sites) and therrs gene (seven mutation sites). We have developed an automated simultaneous detection system of multiple mutations based on thermal dissociation curve analysis for streptomycin resistance inM. tuberculosis using streptavidin-labeled bacterial magnetic particles (SA-BacMPs). With consideration for time and cost effectiveness, we used fewer PCR reactions, with a long PCR target (rpsL, 182 bp;rrs, 467 bp) including multiple mutation sites. In order to improve the amount of target DNA captured on BacMPs through streptavidin-biotin binding, several reaction conditions, such as salt species and concentration in the buffer, and reaction temperature were examined. Compared to the commonly used 1M NaCl solution, the amount of DNA captured on SA-BacMPs was about six times greater (approx 5 pmoles/50 μg BacMPs) in the 2M LiCl solution. Under these conditions, automated nucleotide discriminations of 10 targets inrpsL andrrs genes of streptomycin-resistant and wild-type strains were successfully performed at the same time.     

Genomic rearrangements in a mouse cell line containing integrated SV40 DNA

In the SV40-transformed mouse embryo fibroblast cell line SVT2/S, genomic rearrangements involving the SV40 DNA and flanking host sequences were identified by Southern blot hybridization using viral DNA as probe. No rearrangements of SV40 DNA integrated into nonpermissive mouse cells have been previously described. The standard arrangement found in the majority of subclones was mapped with 20 restriction enzymes, 10 of which cleave sites within the SV40 DNA. A single copy of a defective integrated viral genome is present, in which the late region is missing from about nucleotide 200 clockwise to about nucleotide 1750. The rest of the viral genome including the origin of replication and T antigen binding region is present and colinear with SV40 DNA, except for an internal repeat of about 1750 bp located between nucleotides 2750 and 4500. Rearrangements were found in 4 out of 20 random subclones of the parental SVT2/S cell line and 3 of the 4 continued to rearrange. The thioguanine-resistant cell line 281-1-4, derived from SVT2/S, remained stable on subculture but a chloramphenicol-resistant mutant, 107-6-4, derived from 281-1-4, was highly unstable. In 107-6-4, unique rearrangements were found in 6 of 31 subclones of a population that had undergone abut 25 doublings from a single-cell isolate. The high rate of rearrangement and the sporadic expression of rearrangement potential are characteristic of the transposable controlling elements discovered by McClintock.     

Development of a mouse cell line containing the PhiX174 am3 allele as a target for detecting mutation.

Transgenic mice containing multiple copies of the PhiX174 am3 allele are being developed as a model for detecting tissue-specific in vivo mutation. In order to derive an analogous system for measuring am3 mutation in vitro, cells were cultured from 15-day-old C57Bl/6J mouse embryos that were homozygous for the transgene and these cells were transfected with a plasmid expressing the SV40 large T-antigen. Two G418-resistant colonies were isolated from this culture and expanded to continuously proliferating cell lines (PX-1 and PX-2). Line PX-2 was treated with up to 1.0 mg/ml of N-ethyl-N-nitrosourea (ENU), assayed for survival by cloning efficiency after overnight culture, and assayed for am3 mutations after 5 days of culture. Survival decreased to 31% at the highest dose of ENU, while mutant frequency increased with dose from approximately 2 x 10(-7) in the untreated cells to 13 x 10(-7) in cultures treated with 0.6 mg/ml of ENU. PX-2 cells also were treated with 0 and 0.6 mg/ml of ENU and mutant frequency assays were performed after 5, 24, 48 and 72 h of growth. The mutant frequency in the treated culture increased to 20 x 10(-7) at 48 h and remained approximately the same at 72 h. These results indicate that PX-2 cells should be a useful resource for developing the in vivo am3 mutant assay and for evaluating the sensitivity of the am3 allele to various classes of mutagens.     

Mutagen sensitivity and suppression of position-effect variegation result from mutations in mus209, the Drosophila gene encoding PCNA.

The mus209B1 mutant of Drosophila melanogaster exhibits a complex pleiotropy of temperature-sensitive (ts) lethality, hypersensitivity to DNA-damaging agents such as ionizing radiation and methyl methanesulfonate, suppression of position-effect variegation (PEV), and female sterility. Our discovery that mus209 encodes proliferating cell nuclear antigen (PCNA), which is an indispensable component of the DNA replication apparatus, suggests that alterations to chromosome replication may underlie that pleiotropy. Nine lethal mutations, three of them ts, genetically define the Pcna locus. Temperature shift studies reveal that the vital function of PCNA is required throughout virtually all stages of fly development, and that maternally encoded PCNA is essential for embryogenesis. All three ts mutants strongly suppress PEV, which suggests a role for PCNA in chromatin assembly or modification.     

Maturation-associated increase in IP3 receptor type 1: role in conferring increased IP3 sensitivity and Ca2+ oscillatory behavior in mouse eggs

Maturation of mouse oocytes is accompanied by an increase in sensitivity to inositol 1,4,5-trisphosphate (IP(3))-mediated release of intracellular calcium. To test the hypothesis that the maturation-associated 1.5- to 2.0-fold increase in the mass of the type 1 IP(3) receptor (IP(3)R-1) confers this increase in IP(3) sensitivity, we employed RNA interference to prevent this change in IP(3)R-1 protein level. Microinjection into germinal vesicle (GV)-intact oocytes of dsRNA corresponding to the IP(3)R-1 sequence resulted in a >90% reduction in the amount of maternal IP(3)R-1 mRNA and prevented the maturation-associated increase in the mass of the IP(3)R-1 protein. These injected oocytes matured to metaphase II, and there was no effect on the maturation-associated increases in p34(cdc2)/cyclin B kinase and MAP kinase activities or the global pattern of protein synthesis. IP(3)-induced cortical granule exocytosis was significantly decreased in these eggs when compared with controls previously injected with enhanced green fluorescent protein (EGFP) dsRNA. Following insemination, the IP(3)R-1 dsRNA-injected eggs displayed significantly fewer Ca(2+) transients than controls, and the duration of the first Ca(2+) transient was about half that of controls. These results support the hypothesis that the maturation-associated increase in the mass of IP(3)R-1 confers the increase in IP(3)-sensitivity that is observed following oocyte maturation and is necessary for the proper Ca(2+) oscillatory pattern following insemination.     

Mutagen sensitivity of human lymphoblastoid cells with a BRCA1 mutation in comparison to ataxia telangiectasia heterozygote cells

Our previous results indicated a close relationship between the presence of a BRCA1 mutation in lymphocytes and hypersensitivity for the induction of micronuclei by gamma irradiation and hydrogen peroxide (H(2)O(2)). Comparative investigations with the comet assay (single-cell gel electrophoresis) suggested a normal rate of damage removal and pointed to a disturbed fidelity of DNA repair as a direct or indirect consequence of a BRCA1 mutation. We now wanted to see whether similar results could be obtained with lymphoblastoid cell lines (LCLs) and whether such permanent cells are suitable as a model for the investigation of mechanisms involved in mutagen sensitivity. Our results show that LCLs with a BRCA1 mutation are also hypersensitive to the chromosome-damaging effects of gamma irradiation or H(2)O(2), as revealed by the micronucleus test. Interestingly, LCLs heterozygous for an ataxia telangiectasia (AT) mutation have similar characteristics as BRCA1 cells with respect to the induction and repair of DNA damage induced by either gamma irradiation or H(2)O(2). However, caffeine enhanced the induction of micronuclei by gamma irradiation only in normal and heterozygous AT cells but not in BRCA1 cells, thus indicating a difference in the pathways leading to mutagen sensitivity in cells with a BRCA1 or an AT mutation. Our results suggest that caffeine could be useful in discriminating AT heterozygotes from carriers of a BRCA1 mutation, as well as BRCA1 mutation carriers from normal individuals.     

Genome-wide association for methamphetamine sensitivity in an advanced intercross mouse line

Sensitivity to the locomotor stimulant effects of methamphetamine (MA) is a heritable trait that utilizes neurocircuitry also associated with the rewarding effects of drugs. We used the power of a C57BL/6J × DBA/2J F(2) intercross (n = 676) and the precision of a C57BL/6J × DBA/2J F(8) advanced intercross line (Aap: B6, D2-G8; or F(8) AIL; n = 552) to identify and narrow quantitative trait loci (QTLs) associated with sensitivity to the locomotor stimulant effects of MA. We used the program QTLRel to simultaneously map QTL in the F(2) and F(8) AIL mice. We identified six genome-wide significant QTLs associated with locomotor activity at baseline and seven genome-wide significant QTLs associated with MA-induced locomotor activation. The average per cent decrease in QTL width between the F(2) and the integrated analysis was 65%. Additionally, these QTLs showed a distinct temporal specificity within each session that allowed us to further refine their locations, and identify one QTL with a 1.8-LOD support interval of 1.47 Mb. Next, we utilized publicly available bioinformatics resources to exploit strain-specific sequence data and strain- and region-specific expression data to identify candidate genes. These results illustrate the power of AILs in conjunction with sequence and gene expression data to investigate the genetic underpinnings of behavioral and other traits.     

Tunicamycin-resistant mutations in mouse FM3A cells

Tunicamycin is an antibiotic that inhibits the oligosaccharide synthesis of glycoproteins. It greatly suppressed the growth of cultured mouse mammary carcinoma FM3A cells, when added to growth medium at concentrations of more than 0.1 μg/ml. We have developed a single-step selection system for quantitatively detecting mutations resistant to the antibiotic in FM3A cells. Mutant colonies resistant to 1–1.2 μg tunicamycin per ml (the optimal concentration of the selecting agent) appeared at a frequency of 10⁻⁴ to 10⁻⁵ in an unmutagenized population, but they increased over 50-fold in the population mutagenized with 0.5 μg N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) per ml for 2 h and selected under optimal conditions for the time of mutation expression and cell density in selective medium. Fluctuation analysis, by the method of Luria and Delbrück, revealed that tunicamycin-resistant mutations occurred at random during proliferation in normal medium at a rate of 1.2 × 10⁻⁶ per cell per generation. So far 45 spontaneous and MNNG-induced mutant lines have been isolated and serially passaged in the absence of tunicamycin. These mutant lines all inherited their resistance for more than 60 generations. The mutants examined in detail were 12- to 26-fold more resistant than wild-type cells in terms of the D₁₀ value, the concentration of tunicamycin reducing the plating efficiency to 10% of the control. In the hybrids between wild-type and mutant cells the tunicamycin resistance behaved in a co-dominant manner. Tunicamycin inhibited the incorporation of [³H]mannose into the acid-insoluble cell fraction; in this respect, mutant cells were over 30-fold more resistant than wild-type cells. Possible mechanisms of tunicamycin resistance are discussed.     

Behaviour of mouse macrophage cell line and mouse fibroblast on copolymers containing cellulose triacetate.

Copolymers containing cellulose triacetate were prepared under various conditions. A bacteriological plastic dish was coated with the copolymer (HCTA-MDI copolymer) composed of hydrolysed cellulose triacetate (HCTA) and diphenylmethane diisocyanate (MDI). The deacetylated copolymer (DA-copolymer) was prepared by the deacetylation of HCTA-MDI copolymer. The behaviour of animal cells such as a mouse macrophage cell line (A640-BB-2 cells) and a mouse fibroblast (3T6 cells) on prepared dishes was investigated morphologically. A640-BB-2 cells showed good adhesion and smooth spreading, and 3T6 cells also showed good adhesion and sufficient cell growth on the dish coated with HCTA-MDI copolymer. These results suggest that these copolymers are useful for biomedical materials.     

Using embryonic stem cells to introduce mutations into the mouse germ line

It is now possible, through the use of a number of experimental technologies, to transfer genetic information into mouse embryos to stably alter the genetic constitution of mice. This experimental approach, namely the generation of so-termed "transgenic" animals, is affording new insights into a wide variety of biological problems. This review focuses on one system for the generation of transgenic mice, which utilizes tissue culture cell lines of embryonic stem cells, termed ES cells. The remarkable property of ES cells is that they retain the potential to reform an embryo; when they are replaced inside a carrier embryo, they resume normal development and contribute to all the tissues of the live-born chimeric animal. Recent experiments, using a repertoire of gene transfer techniques, have shown that ES cells are amenable to a variety of experimental manipulations in tissue culture. Moreover, it has been demonstrated that these genetically altered cells can be transferred into the germ line of chimeric mice, thus allowing the production of unique strains of animals for study. The applications of the ES cell system are reviewed, with particular emphasis on their use for the generation of random insertional mutations using a retrovirally mediated mutagenesis approach. Finally, the use of ES cells in conjunction with the recently described technique of homologous recombination, or "gene targeting," is discussed. This technology allows the generation of animals carrying extremely precise genetic modifications of endogenous genes.     

Isolation and characterization of a mouse cell line containing a defective Moloney murine leukemia virus genome.

A culture of mouse cells containing a 1,000-nucleotide deletion mutant of Moloney murine leukemia virus has been isolated. The deletion did not affect the size or function of the 21S mRNA that encodes the env gene products. Both the deleted RNA and the 21S mRNA were recovered in polyribosomes. Cells containing the deleted virus made no detectable Pr180gag-pol. Pr65gag synthesis with also absent, but a 45,000-molecular-weight gag gene product was found that might be encoded by the deleted genome. Biosynthesis of Pr80env proceeded normally in these cells; the intracellular precursor was cleaved and migrated to the cell surface as gp70. The cells could not be superinfected by homologous Moloney murine leukemia virus presumably because of surface restriction due to the gp70. Although the cells express the Moloney murine leukemia virus gp70 on their surface, they will not make pseudotypes after infection with vesicular stomatitis virus implying that Pr65gag may play a critical role in pseudotype formation. Induction of endogenous virus expression in the cells carrying the deletion mutant generated an N-tropic murine leukemia virus that can fuse XC cells. This may represent a recombinant between the deletion mutant and an endogenous virus.