ENU induced mutations causing congenital cardiovascular anomalies

We used non-invasive high frequency ultrasound to screen N-ethyl-N-nitrosourea mutagenized mouse fetuses for congenital cardiovascular anomalies. We ultrasound scanned 7546 mouse fetuses from 262 mutagenized families, and identified 124 families with cardiovascular defects. Represented were most of the major congenital cardiovascular anomalies seen clinically. The ENU-induced mutations in several families were mapped using polymorphic microsatellite DNA markers. One family with forelimb anomalies and ventricular septal defects, phenotypes similar to Holt-Oram syndrome, and one family with transposition of the great arteries and heart situs anomalies were mapped to different regions of mouse chromosome 4. A third mutation causing persistent truncus arteriosus and craniofacial defects, phenotypes reminiscent of DiGeorge syndrome, was mapped to mouse chromosome 2. We note that mouse chromosomes 4 and 2 do not contain Tbx5 or Tbx1, genes previously linked to Holt-Oram and DiGeorge syndromes, respectively. In two other families, the ENU-induced mutation was identified--Sema3CL605P was associated with persistent truncus arteriosus with interrupted aortic arch, and the Gja1W45X connexin43 mutation caused conotruncal malformation and coronary aneurysms. Although our screen was designed as a recessive screen, a number of the mutations showed cardiovascular phenotypes in both heterozygote and homozygote animals. These studies show the efficacy of ENU mutagenesis and high-throughput ultrasound phenotyping in recovering mutations causing a wide spectrum of congenital heart defects. These ENU-induced mutations hold promise in yielding new insights into the genetic basis for human congenital heart disease.     

Contrasting effects of VEGF gene disruption in embryonic stem cell-derived versus oncogene-induced tumors

Previous gene targeting studies have implicated an indispensable role of vascular endothelial growth factor (VEGF) in tumor angiogenesis, particularly in tumors of embryonal or endocrine origin. In contrast, we report here that transformation of VEGF-deficient adult fibroblasts (MDF528) with ras or neu oncogenes gives rise to highly tumorigenic and angiogenic fibrosarcomas. These aggressive VEGF-null tumors (528ras, 528neu) originated from VEGF(-/-) embryonic stem cells, which themselves were tumorigenically deficient. We also report that VEGF production by tumor stroma has a modest role in oncogene-driven tumor angiogenesis. Both ras and neu oncogenes down-regulated at least two endogenous inhibitors of angiogenesis [pigment epithelium derived factor (PEDF) and thrombospondin 1 (TSP-1)]. This is functionally important as administration of an antiangiogenic TSP-1 peptide (ABT-526) markedly inhibited growth of VEGF(-/-) tumors, with some ingress of pericytes. These results provide the first definitive genetic demonstration of the dispensability of tumor cell-derived VEGF in certain cases of 'adult' tumor angiogenesis, and thus highlight the importance of considering VEGF-independent as well as VEGF-dependent pathways when attempting to block this process pharmacologically.     

Conditionally lethal amber mutations in the leader peptidase gene of Escherichia coli.

The lep gene of Escherichia coli encodes the leader peptidase which cleaves amino-terminal leader sequences of secreted proteins. To facilitate the study of structure-function relationships of the leader peptidase, 22 amber mutations in lep were isolated by localized mutagenesis. These amber mutants grew at 32 degrees C but not at 42 degrees C in the presence of a temperature-sensitive amber suppressor. Most of them were lethal under sup0 conditions. However, one amber mutant, the lep-9 mutant, exhibited temperature-sensitive growth in the sup0 strain, indicating that the amber fragment is active at 32 degrees C but not at 42 degrees C. Protein precursors of the maltose-binding protein and OmpA accumulate strikingly in the lep-9 mutant.     

Specificity of embryonic lethal mutations in Drosophila analyzed in germ line clones

Summary Only a small fraction of the known mutations causing death to homozygous Drosophila produce gross morphological defects during embryogenesis. We have examined fourteen such loci on the X-chromosome to determine: 1) whether the requirement for their respective activities is restricted to embryogenesis; and 2) whether the embryonic phenotype in mutant embryos is affected by the dosage of wild-type alleles in the mother. For two alleles per locus germ line clones were produced during larval development by irradiating females heterozygous for the lethal mutation and a dominant female sterile (ovo^D). Only one of the 14 loci (armadillo) is required during development of the germ cell to make morphologically normal eggs. Mutations at two other loci, (bazooka and Notch), allow normal oogenesis but cause major reductions in the viability of genetically normal (i.e., heterozygous) progeny. The majority of the loci (11/14) are not required in the germ line for either oogenesis or embryogenesis. However, in three cases (extradenticle, faintoid and lethal myospheroid), germ line homozygosity results in a readily detectible enhancement of embryonic phenotype over that observed in embryos derived from heterozygous mothers still possessing one wild type allele. The same six loci which show the most substantial effects on germ line homozygosity (arm, baz, N, exd, ftd and mys) also show an amelioration of the mutant phenotypes when maternal dosage is increased to wild type levels by using attached-X females. Four of these same loci (arm, baz, N and exd were cell lethal in imaginal discs.     

Dominant lethal mutations in the dnaB helicase gene of Salmonella typhimurium

A class of dominant lethal mutations in the dnaB (replicative helicase) gene of Salmonella typhimurium is described. The mutated genes, when present on multicopy plasmids, interfered with colony formation by Escherichia coli host strains with a functional chromosomal dnaB gene. The lethal phenotype was expressed specifically in supE (glutamine-inserting) host strains and not in Sup+ strains, because the mutant genes, by design, also possessed an amber mutation derived from a glutamine codon. Mutations located at 11 sites by deletion mapping and DNA sequence analysis varied in the temperature dependence and severity of their lethal effects. None of the mutations complemented a dnaB(Ts) host strain at high temperature (42 degrees C). Therefore, these nonfunctional DnaB proteins must engage some component(s) of the DNA replication machinery and inhibit replication. These mutations are predicted to confer limited, specific defects in either the catalytic activity of DnaB or the ability of DnaB to interact with one of its ligands such as DNA, nucleotide, or another replication protein. The variety of mutant sites and detailed phenotypes represented in this group of mutations may indicate the operation of more than one specific mechanism of lethality.     

A system to efficiently maintain embryonic lethal mutations in the flour beetle Tribolium castaneum

 Due to its small size, short life cycle, and easy maintenance, the flour beetle Tribolium castaneum is well suited for the genetic analysis of development. One drawback of Tribolium as a genetic system is, however, the difficulty of keeping embryonic lethal lines. Presently, only few lethal mutations can be kept as balanced stocks. Therefore, heterozygous carriers must be identified anew in every generation in order to maintain a recessive embryonic mutation. To alleviate this problem we have devised a block system that allows the simultaneous processing of many mutant lines or test crosses for visual inspection of larval cuticle phenotypes. Using this technique, one person can maintain about 100 embryonic lethal stocks, which makes feasible the thorough genetic analysis of embryogenesis in this species. Received: 4 November 1998 / Accepted: 7 February 1999     

ENU induces mutations in the heart of lacZ transgenic mice

The use of transgenic mouse models as somatic mutation assays allows determination of mutation in all tissues of the mouse, including non-dividing tissues. In this regard, these models can be used to study the possibility that mutations can be induced in mitotically quiescent organs such as the heart. Mutations are generally thought to be associated with mitotic processes of DNA replication. Mutations, however, are also postulated to occur in the absence of mitosis as the result of DNA repair. In order to determine whether or not mutations could be induced in the heart, we analyzed the mutant frequency in the hearts of F(1) (Muta Mouse X SWR) mice that had been treated acutely with 250 mg/kg ENU and sampled at days 10, 35, and 70 post-treatment. A significant increase in mutant frequency at day 70 shows that mutations can be induced in the heart. Since the heart contains small numbers of non-muscle cells, additional mechanisms that could explain these results were also considered. The effect of ENU-induced cell proliferation or a sub-population of rapidly dividing cells is ruled out by C(14)-thymidine uptake studies which showed minimal proliferation. By the same token, the influence of ex vivo mutations (i.e., DNA adducts fixed as mutations during replication in the bacteria) is ruled out by the observed time course of mutations, as well as experimental evidence showing that such mutations are not detected in the lacZ assay.     

Selection for mutations in the cDNAs of transgenic mice upon expression of an embryonic lethal protein

The generation of transgenic mouse models to study in vivo functions of specific proteins has become common practice. In addition, PCR technology allows efficient and rapid identification of founder mice by the analysis of tail tip DNA. Whilst the DNA construct used in the microinjection of one-cell-stage embryos is usually sequenced it is not common practice to sequence the PCR product once the transgene has been inserted into the mouse genome. In this report we describe why sequencing of inserted transgenes is important. Upon generation of transgenic mice expressing a splice variant of MDM2, MDM2-A, three of four founders contained mutations within the Mdm2-a cDNA sequence. The observation that selection against expression of wild-type MDM2-A resulted in the generation of mice expressing mutant transgenes highlights the importance of sequencing the transgenes of founder mice.     

Chemical and radiation induced late dominant lethal effects in mice.

Although theoretically expected, experimental data to date have not shown dominant lethal expression to occur throughout the developmental period. Specifically, late post-implantation effects have not been demonstrated. We routinely use an experimental technique in which parental females mated to mutagenically treated males are allowed to give birth and wean their litter, and their uterine horns are then inspected for uterine scars indicative of live and dead embryos. In a number of experiments in which males were mutagenically treated with either chemicals or X-irradiation, a discrepancy was observed between the number of live embryos as determined by the scar technique and the number of live observed at birth, suggesting the possibility of embryonic losses at a late stage in development. Initial analyses showed that mutagenic treatment increased the percentage of these late losses. These differences were statistically significant in 2 of 3 analyses. Factors affecting statistical significance and an understanding of dominant lethal mutations are discussed.     

Lambda transducing phages for the nalA gene of Escherichia coli and conditional lethal nalA mutations.

Summary Defective transducing phages for the nalA region of the Escherichia coli chromosome were isolated from a lysogen in which is inserted in the nearby glpT gene. The three classes of transducing phages designated nrdA, dubiG, and dnalA contained bacterial DNA extending from glpT through nrdA, ubiG, and nalA, respectively. The bacterial genes are in the left arm of the chromosome. Of the eleven polypeptides coded by dnalA that were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate only one was not also specified by dubiG This 105,000 dalton polypeptide is the nalA gene product. The electrophoretic mobility and isoelectric point of this protein were unaffected by a nalA mutation (nalA48) that confers nalidixic acid resistance. Temperature-sensitive and amber mutations in the nalA gene were isolated using a dnalA48 lysogen which is heterodiploid for nalA. The conditional lethality of these mutations proves that nalA is an essential locus.     

Contrasting Effects of ENU Induced Embryonic Lethal Mutations of thequakingGene

Multiple alleles of the quaking (qk) gene have a variety of phenotypes ranging in severity from early embryonic death to viable dysmyelination. A previous study identified a candidate gene, QKI, that contains an RNA-binding domain and encodes at least three protein isoforms (QKI-5, -6 and -7). We have determined the genomic structure of QKI, identifying an additional alternative end in cDNAs. Further we have examined the exons and splice sites for mutations in the lethal allelesqk^l-1, qk^kt1, qk^k2,andqk^kt3.The mutation inqk^l-1creates a splice site in the terminal exon of the QKI-6 isoform. Missense mutations in the KH domain and the QUA1 domains inqk^k2andqk^kt3,respectively, indicate that these domains are of critical functional importance. Although homozygotes for each ENU induced allele die as embryos, their phenotypes as viable compound heterozygotes with qk^vdiffer. Compound heterozygousqk^vanimals carryingqk^kt1, qk^k2,andqk^kt3all exhibit a permanent quaking phenotype similar to that ofqk^v/qk^vanimals, whereasqk^v/qk^l-1animals exhibit only a transient quaking phenotype. Theqk^l-1mutation eliminates the QKI-5 isoform, showing that this isoform plays a crucial role in embryonic survival. The transient quaking phenotype observed inqk^v/qk^l-1mice indicates that the QKI-6 and QKI-7 isoforms function primarily during myelination, but that QKI-5 may have a concentration-dependent role in early myelination. This mutational analysis demonstrates the power of series of alleles to examine the function of complex loci and suggests that additional mutant alleles of quaking could reveal additional functions of this complex gene.     

A genetic screen for zygotic embryonic lethal mutations affecting cuticular morphology in the wasp Nasonia vitripennis

We have screened for zygotic embryonic lethal mutations affecting cuticular morphology in Nasonia vitripennis (Hymenoptera; Chalcidoidea). Our broad goal was to investigate the use of Nasonia for genetically surveying conservation and change in regulatory gene systems, as a means to understand the diversity of developmental strategies that have arisen during the course of evolution. Specifically, we aim to compare anteroposterior patterning gene functions in two long germ band insects, Nasonia and Drosophila. In Nasonia, unfertilized eggs develop as haploid males while fertilized eggs develop as diploid females, so the entire genome can be screened for recessive zygotic mutations by examining the progeny of F1 females. We describe 74 of >100 lines with embryonic cuticular mutant phenotypes, including representatives of coordinate, gap, pair-rule, segment polarity, homeotic, and Polycomb group functions, as well as mutants with novel phenotypes not directly comparable to those of known Drosophila genes. We conclude that Nasonia is a tractable experimental organism for comparative developmental genetic study. The mutants isolated here have begun to outline the extent of conservation and change in the genetic programs controlling embryonic patterning in Nasonia and Drosophila.     

An Arabidopsis embryonic lethal mutant with reduced expression of alanyl—t RNA synthetase gene

In present paper,one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identified and designated as acd mutant.The embryo developmant of this mutant is arrested in globular stage,The cell division pattern is abnormal during early embryogenesis and results in distubed cellular differentiation.Most of mutant embryos are finally degenerated and aborted in globular stage,However,a few of them still can germinate in agar palte and produce seedlings with shoter hypoctyl and distorted shoot meristem.To understand the molecular basis of the phenotype of this mutant,the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening.According to the sequence analysis and similarity searching,a 936 bp cDNA sequence(EMBL accession #:Y12555)from selectoed positive clone shows a 99.8%(923/925bp) sequence homolgy with Alanyl-tRNA Synthetase(AlaRS) gene of Arabidopsis thaliana.Furthermore,the data of in situ hybridization experiment indicate that the expression of Ala RS gene is weak in early embryogenesis and declines along with globular embryodevelopment in this mutant Accordingly,the reduced expression of Ala RS gene may be closely related to the morphological changes in early embryogenesis of this lethal mutant.     

Establishment of embryonic stem cell lines from preimplantation mouse embryos homozygous for lethal mutations in the t-complex

We have determined the frequency at which embryonic stem cell (ESC) lines can be established from inner cell masses (ICMs) isolated from blastocysts homozygous for lethal mutations in the mouse t-complex. Approximately one-third of the expected number, 3/29, of the ESC lines established from embryos obtained by inter-se mating of +/tw18 mice are homozygous for the tw18 haplotype. These tw18/tw18 ESC lines form a variety of cell types in vitro and in vivo, including mesodermal derivatives such as cartilage and muscle. On the basis of these and data from other studies, we suggest that the normal function of the gene represented by the tw18 lethal allele is required for multiplication/survival of mesodermal precursors in the embryo rather than the specification of the mesodermal lineage, and that the lethal effects of this mutation are expressed in only the highly structured environment of the early postimplantation embryo. In studies of the lethal tw5 haplotype, we found that 2/2 ESC lines obtained are mutant homozygotes. Analysis of these data, in conjunction with the results of our earlier study (Magnuson, T., Epstein, C. J., Silver, L. M., and Martin, G. R. (1982), Nature (London) 298, 750-753), suggests that homozygosity for the genes found in the tw5 haplotype does not reduce cell viability. By contrast, 0/16 ESC lines isolated from embryos obtained from matings of +/t0 mice are mutant homozygotes. Analysis of the genotypes of ICM-derived primary stem cell colonies suggests that t0 homozygous ICM cells are unable to undergo sufficient proliferation in vitro to give rise to ESC lines.     

X-ray induced dominant lethal mutations in mature and immature oocytes of guinea-pigs and golden hamsters.

The induction of dominant lethal mutations by doses of 100-400 rad X-rays in oocytes of the guinea-pig and golden hamster was studied using criteria of embryonic mortality. For both species higher yields were obtained from mature than from immature oocytes, in contrast to results for the mouse. Data on fertility indicated that in the golden hamster, as in the mouse, immature oocytes were more sensitive to killing by X-rays than mature oocytes but that the converse was true in the guinea-pig. The dose-response relationship for mutation to dominant lethals in pre-ovulatory oocytes of guinea-pig and golden hamsters was linear, both when based on pre- and post-implantation loss and when on post-implantation loss only. The rate per unit dose was higher for the golden hamster, and the old golden hamsters were possibly slightly more sensitive than young ones. The mutation rate data for mature oocytes of the mouse, using post-implantation loss alone, also fitted a linear dose-response relationship, except that the rate per unit dose was lower than for the other two species.