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PubMed1979

Extrachromosomal control of mutability in Drosophila melanogaster.

Hybrid dysgenesis is a syndrome of germ-line aberrations including, e.g., sterility and mutation, found in certain interstrain hybrids of Drosophila melanogaster. Previous studies of sterility have shown that elements responsible for dysgenesis may reside on all major chromosomes, but that their dysgenesis-causing ability is controlled by an unknown extrachromosomal factor. Dysgenic hybrids also give rise to unstable visible mutations thought to be DNA insertions at certain sensitive loci. One such unstable allele at the singed bristle locus, designated snw, was found to mutate at extraordinary rates exceeding 50%. This instability was shown to be under the same extrachromosomal control as hybrid dysgenesis itself. That is, the mutability of snw was reversibly suppressed when placed in the background cytotype known to prevent sterility and other characteristics of hybrid dysgenesis. These results suggest that snw may represent an insertion at the singed locus of a hypothetical gene responsible for hybrid dysgenesis.

Animals↗
PubMed1992

Studies on the genotoxicity of monocrotophos in somatic and germ-like cells of Drosophila.

Parryfos, a farm-grade formulation of monocrotophos, was tested for genotoxicity in the wing primordial cells and the male germ-line cells of Drosophila melanogaster. Larvae of the 2nd or 3rd instar, heterozygous for the wing-cell marker mutations mwh and flr3, were exposed to different concentrations of the insecticide in the food. The wings of the hatched flies were screened for the presence of mutant mosaic spots exhibiting the marker phenotypes. The frequency of induction of sex-linked recessive lethal mutations was used to assess genotoxic effects in male germ-line cells. The tested compound was genotoxic in both the somatic and the germ-line cells of Drosophila.

Animals↗
PubMedDate not supplied

The repertoire of antibodies to a hapten group in an inbred strain of mouse.

We have investigated the diversity of antibodies to the p-azophenylarsonate (Ar) hapten group in the A strain of mouse. The method used consisted of suppressing an idiotype which commonly is observed in the anti-Ar antibodies of all this strain, and then generating anti-idiotypic antisera against the anti-Ar antibodies (lacking the cross-reactive idiotype) which arose after hyperimmunization of the suppressed mice. Anti-idiotypic antibodies prepared against these anti-Ar antibodies were used as a tool to investigate the frequency of recurrence of individual idiotypes in other mice of the same strain. A highly sensitive radioimmunoassay indicated that 3 of the 4 idiotypes studied were virtually undetectable in a panel of 181 A/J mice immunized against the phenylarsonate hapten. A fourth idiotype was found but at very low frequency. The results emphasize the enormous diversity of the immune response to a single hapten group within a strain of mouse. They are interpreted on the basis of hypothesis that the diversity arises through a random series of somatic events. The common cross-reactive idiotype, which is present in the anti-Ar antibodies of all normal A/J mice, is postulated to be the product of a germ-line gene which is closely related to a germ-line gene through a small number of somatic mutations which takes place in every A/J mouse.

Animals↗
PubMed1976

Totipotency and normal differentiation of single teratocarcinoma cells cloned by injection into blastocysts.

A definitive test for developmental totipotency of mouse malignant teratocarcinoma cells was conducted by cloning singly injected cells in genetically marked blastocysts. Totipotency was conclusively shown in an adult mosaic female whose tumor-strain cells had made substantial contributions to all of the wide range of its somatic tissues analyzed; the clonally propagated cell lineage had therefore differentiated in numerous normal directions. The test cells were from "cores" of embryoid bodies of a euploid, chromosomally male (X/Y), ascites tumor grown only in vivo by transplantation for 8 years. The capacity of cells from the same source to differentiate, in a phenotypic male, into reproductively functional sperms, has been shown in our previous experiments [(1975) Proc. Nat. Acad. Sci. USA 72, 3585-3589]. Cells from this transplant line therefore provide material suitable for projected somatic and germ-line genetic analyses of mammalian differentiation based on "cycling" of mutation-carrying tumor cells through developing embryos. In some animals obtained from single-cell injections tumor-derived cells were sporadically distributed in developmentally unrelated tissues. These cases can be accounted for by delayed and haphazard cellular integration, and by a marked degree of sustained cellular developmental flexibility in early mammalian development, irrespective of certain classical "germ-layer" designations. All mosaic mice obtained have thus far been free of teratomas. In one case, the injected stem cell contributed only to the pancreas and gave rise to a malignancy resembling pancreatic adenocarcinoma. The high modal frequency of euploidy in these individually tested cells thus tends to indicate that a near-normal chromosome complement is sufficient for total restoration of orderly gene expression in a normal embryonic environment; it may also be necessary for teratoma stem-cell proliferation to be terminated there.

Alleles↗
PubMed1992

The unc-18 gene encodes a novel protein affecting the kinetics of acetylcholine metabolism in the nematode Caenorhabditis elegans.

Genes affecting acetylcholine (ACh) levels without influencing choline acetyltransferase activity have been identified in Caenorhabditis elegans. We have examined one such gene, unc-18. We isolated a transposon-insertion allele for unc-18 and used it to clone a genomic region containing the unc-18 locus. The unc-18 location within this region was determined by rescuing the unc-18 mutant phenotype in a germ-line transformation experiment and identifying transcripts affected by four independent unc-18 mutations. A single-sized poly(A)+ RNA was synthesized from the gene. Expression of the transcript appears to be stage specific: The transcript is found in abundance at the early larval stage but in decreased amounts at the fourth larval and the adult stages. These results show that the unc-18 gene plays a role in development as well as in the kinetics of ACh metabolism.

Acetylcholine↗
PubMed1979

Evidence supporting somatic assembly of the DNA segments (minigenes), coding for the framework, and complementarity-determining segments of immunoglobulin variable regions.

Two sets of apparently conflicting data on the genes coding for the variable region are being accumulated. One suggests that the sets of nucleotides coding for the framework segments of immunoglobulin light and heavy (VL and VH) chains assort independently and are therefore germ-line minigenes which, together with sets of nucleotides coding for the complementarity-determining regions (CDR) or segments assemble to form complete variable (V)-region genes (15, 16, 33). The other, based on the findings with clones from 12-d-old embryo and adult mouse coding for V-regions, infer that the first three frameworks and the three complementarity-determining segments are already assembled as germ-line V-genes (17-21). It is now generally accepted that the J segment, which in the one instance sequenced (21) is made up of nucleotides coding for framework (FR)4 plus two residues of CDR3, is a minigene. An examination of sequences of human, mouse, and rabbit V-regions, assuming the latter hypothesis, indicates that individual framework sets would have to be present in many copies. The FR2 segment found in one human, 20 mice, and 13 rabbits would have to be present in at least 10/14 copies in the NZB, and 5/6 in the BALB/c mouse, and 12/13 in the rabbit. The X-ray crystallographic data show this region to be a loop, projecting out from the V-domain, capable of accommodating many substiutions and 12 and 8 alternative sequences for this FR2 segment have been found in mouse and rabbit VK chains with substitutions possible at 13 of the 15 positions. These alternative sequences occur much less frequently than the preserved FR2 segment. Thus, there is no basis in the protein structure to account for evolutionary stability of this FR2 segment if it occurs in so many copies in germ-line genes coding for residues 1-96, but its stability is easily explained if it were coded for by a separate germ-line minigene present as a single copy; the alternative forms could then have arisen by duplication and mutation of this minigene. Somatic assembly of the minigene segments for the three framework and three complementarity-determining segments during differentiation would account completely for our assortment data from which FR4 was inferred to be a minigene.

Amino Acid Sequence↗
PubMedDate not supplied

B-cell ontogeny in the chicken.

We conclude that our data are most consistent with the hypothesis that individual bursal stem cells give rise to multiple clones of B lymphocytes by a preprogrammed sequence of variable (V) region genes. The alternate hypothesis that each stem cell gives rise to one B cell expressing but one set of VH genes could only fit our data if the additional assumption is made that each stem cell is preprogrammed to give rise to an unique B-cell clonotype at a fixed time during development. Since we were unable to influence the pattern of expression of clonotypic diversity by modifying the exposure to antigens and since random somatic mutations would seem an inefficient mechanism for systematic generation of the diversity, we favor the possibility of a genetic mechanism involving an orderly pairing of germ-line VH and VL genes. The corollary of this hypothesis is that V-region diversity is generated first and Ig-class diversity is secondarily expressed within each clone by a switch mechanism for the sequential expression of CH genes.

Animals↗
PubMed1992

Polymorphism of human immunoglobulin VH4 germ-line genes.

The human immunoglobulin VH4 gene family is thought to contain approximately 10 germ-line genes and to exhibit little polymorphism. We report here an analysis of VH4 germ-line genes that were amplified from DNA of two unrelated individuals. Ten unique (non-repetitive) sequences were obtained from individual A and 11 from individual B. Nine of these sequences represent new germ-line genes, and 8/9 exhibit only 89%-96% similarity to genes identified previously. Subsets of VH4 genes displayed distinctive nucleotide motifs that account for most of the differences between them. This observation suggests that diversity in the VH4 gene family arose from the acquisition of blocks of nucleotides, rather than by accumulation of point mutations. These nucleotide blocks could have been acquired by gene conversion or by homologous recombination. All of the VH4 genes have a potential N-linked glycosylation site at Asn 60, and some genes encode a second site at Asn 52. The VH4 gene family is larger and more polymorphic than appreciated previously. Immunoglobulin gene polymorphism may make a significant contribution to hereditary variations in the immune response and to the genetic predisposition to autoimmune diseases.

Amino Acid Sequence↗
PubMed1992

Cystic fibrosis in the mouse by targeted insertional mutagenesis.

Cystic fibrosis is a fatal genetic disorder which afflicts 50,000 people worldwide. A viable animal model would be invaluable for investigating and combating this disease. The mouse cystic fibrosis transmembrane conductance regulator gene was disrupted in embryonal stem cells using an insertional gene targeting vector. Germ-line chimaeras were derived and the offspring of heterozygous crosses studied. These homozygous mutant mice survive beyond weaning. In vivo electrophysiology demonstrates the predicted defect in chloride ion transport in these mice and can distinguish between each genotype. Histological analysis detects important hallmarks of human disease pathology, including abnormalities of the colon, lung and vas deferens. This insertional mouse mutation provides a valid model system for the development and testing of therapies for cystic fibrosis patients.

Animals↗
PubMed1975

Normal genetically mosaic mice produced from malignant teratocarcinoma cells.

Malignant mouse teratocarcinoma (or embryonal carcinoma) cells with a normal modal chromosome number were taken from the "cores" of embryoid bodies grown only in vivo as an ascites tumor for 8 years, and were injected into blastocysts bearing many genetic markers, in order to test the developmental capacities, genetic constitution, and reversibility of malignancy of the core cells. Ninety-three live normal pre- and postnatal animals were obtained. Of 14 thus far analyzed, three were cellular genetic mosaics with substantial contributions of tumor-derived cells in many developmentally unrelated tissues, including some never seen in the solid tumors that form in transplant hosts. The tissues functioned normally and synthesized their specific products (e.g., immunoglobulins, adult hemoglobin, liver proteins) coded for by strain-type alleles at known loci. In addition, a tumor-contributed color gene, steel, not previously known to be present in the carcinoma cells, was detected from the coat phenotype. Cells derived from the carcinoma, which is of X/Y sex chromosome constitution, also contributed to the germ line and formed reproductively functional sperms, some of which transmitted the steel gene to the progeny. Thus, after almost 200 transplant generations as a highly malignant tumor, embryoid body core cells appear to be developmentally totipotent and able to express, in an orderly sequence in differentiation of somatic and germ-line tissues, many genes hitherto silent in the tumor of origin. This experimental system of "cycling" teratocarcinoma core cells through mice, in conjunction with experimental mutagenesis of those cells, may therefore provide a new and useful tool for biochemical, developmental, and genetic analyses of mammalian differentiation. The results also furnish an unequivocal example in animals of a non-mutational basis for transformation to malignancy and of reversal to normalcy. The origin of this tumor from a disorganized embryo suggests that malignancies of some other, more specialized, stem cells might arise comparably through tissue disorganization, leading to developmental aberrations of gene expression rather than changes in gene structure.

Animals↗
PubMed1992

Molecular analysis of the V kappa III-J kappa junctional diversity of polyclonal rheumatoid factors during rheumatoid arthritis frequently reveals N addition.

Much interest was stirred in recent years by the evidence that rheumatoid factors (RF) variable regions are encoded by a restricted set of V genes, with little or no somatic mutations, that are often overexpressed in the fetal repertoire. This is reminiscent of what has been observed for natural autoantibodies. However, these data come from studies of monoclonal RF (mRF) isolated from patients with lymphoproliferative disorders who usually do not present autoimmune symptoms. The molecular characterization of RF during autoimmune diseases such as rheumatoid arthritis (RA) has been hampered for some time because of their polyclonality; recently using the polymerase chain reaction method, we have demonstrated that RF kappa variable regions from a patient with RA were encoded by V kappa III genes known to code for mRF but that these genes had undergone somatic mutations with a pattern suggesting an antigen-driven maturation. Because an important role of the light chain third complementarity-determining region (CDR3) in anti-IgG reactivity and idiotype expression has already been suspected for RF, we now report the molecular characterization of the junction regions of these rearranged V kappa gens. Surprisingly, our data show that in 55% of the cases there is addition of a proline and/or glycine amino acid residue at the recombination site between V kappa and J kappa. The sequence analysis of our patients' germ-line Vg and J kappa 4 genes segments and their flanking regions demonstrates that the additional codons are not readily explicable by recombination between germ-line sequences and probably result from an N addition process. Since we could not find such an additional codon in 15 previously published mRF kappa chains we suggest that "pathogenic" RF during RA and mRF derive from different, although overlapping, B cell subsets. Moreover, since additional codons at the recombination site of V kappa and J kappa seem exceptional in expressed human kappa chains and because the resulting amino acid residue is a proline in most cases, we think that RF kappa chain CDR3 is under a very strong selective pressure during RA.

Amino Acid Sequence↗
PubMedDate not supplied

The complete sequence of the murine monoclonal immunoglobulin MOPC 173 (IgG2a): genetic implications.

The complete amino acid sequence of the murine monoclonal immunoglobulin MOPC 173 (IgG2a, kappa) is reported. The heavy chain contains 447 amino-acid residues, and one carbohydrate prosthetic group attached to the ASX residue 299. The kappa light chain is composed of 214 residues. The H chains are covalently linked by 3 interchain disulfide bridges. The H-L bond-forming cysteine of the H chain is between the VH and the CH1 domain. Intrachain bridges are disposed linearly, according to the classical model. There is no simple relationship between the primary structure and any given function of a particular domain. This is presumably due to the fact that the selection pressure exerts itself on the three-dimensional structure which may retain a conserved general organization as a result of balanced multiple mutations. Selection seems to act in two ways: --horizontally, in a multigene system such as the immunoglobulin classes (C domains of the heavy chains), leading to interclass homologies which are particularly marked for all the COOH-terminal domains of H and L chains which have, in addition a fair degree of homology with human beta2 microglobulin (about 30% identities); --vertically, in which case strictly homologous domains appear extremely conserved between distinct animal species. Conservation of the VH domains seems just as high as conservation of the CH domains. The VH region contains 3 types of positions: invariant and subgroup characteristic ("framework") which may be accounted for by a rather small number of germ-line genes, and hypervariable for which the origin of diversity, somatic or germinal, cannot be decided from sequence data alone. Murine VK domains, although basically built according to the same pattern, show a much more marked polymorphism of the framework, which might necessitate a higher number of basic germ-line genes. Finally, a hypothetical model of the switch mechanism is proposed. Rotational symmetry regions can be deduced at the DNA level from the known amino acid sequences of the switch peptides for the three translocational systems: H, kappa and lambda. These would provide recognition signals for restriction-like enzymes such as those which operate in prokaryotes. An implication of this model is the definition of an exact limit between the V and the C regions of all immunoglobulin chains.

Amino Acid Sequence↗
PubMed1992

The forces driving autoimmune disease.

There are two classes of autoimmune disease, organ-specific and non-organ specific or systemic. That cells producing autoantibodies are selected by antigen is strongly suggested by the presence of mutations and high affinity antibody. T-cells are pivotal in all forms of autoimmunity as evidenced by the therapeutic benefit of anti-T-cell monoclonals such as anti-CD4, and the frequent development of high affinity IgG autoantibodies. The production of anergic T-cells by the use of non-depleting anti-CD4 in the presence of antigen is discussed with particular reference to its potential for immunological intervention in autoimmune disease. It is possible to identify T-cell epitopes in organ-specific autoimmunity using pathogenic T-cell clones or hybridomas to identify the peptide sequences which are reactive. Antigen-specific therapy may ultimately be based on such peptide epitopes. The specificity of the T-cells in systemic autoimmunity is still obscure, but there is some evidence that reactivity with certain germ-line idiotypes can lead to the development of systemic autoimmunity. The possibility of stimulating B-cells specific for auto-antigens such as DNA becomes feasible if a complex of antibody and DNA is taken up by these specific B-cells and processed idiotype is presented to T-helpers specific for those idiotype epitopes. Evidence is presented that there may be pre-existing defects in the target organ in certain organ-specific disorders, and the evidence for a glycosylation defect in the IgG in patients with rheumatoid arthritis is explored. It is noted that the spouses of probands with rheumatoid arthritis is explored. It is noted that the spouses of probands with rheumatoid arthritis also tend to have this glycosylation defect and this raises the possibility of an effect due to an environmental factor, such as a microbial infection. Molecular mimicry of autoantigens by microbes can stimulate autoreactive cells by their cross-reactivity. It is emphasized that cross-reaction which gives rise to the priming of autoreactive T-cells could give rise to the establishment of a chronic autoimmune state. In animals with normal regulatory immune systems, such induced autoimmunity is ultimately corrected and it is only in animals where there are defects in regulation, that autoimmunity persists. Thus, there are many factors giving rise to autoimmunity, and the diseases are rightly regarded as multifactorial in origin.

Amino Acid Sequence↗
PubMed1992

Chorion gene cis-regulatory DNA restricts tissue specificity of reporter gene expression in transformed Drosophila.

P element mediated germ-line transformation was used to study the developmental specificity of Drosophila chorion gene regulatory sequences directing expression of the bacterial reporter genes for chloramphenicol acetyltransferase (CAT) and beta-galactosidase (lacZ). DNA fragments containing 5' flanking plus the entire 5' untranslated and the beginning of the coding region of either the s36 or the s15 chorion gene are able to confer on the reporter genes normal tissue as well as temporal specificity of expression, exclusively in the ovary of transformed female flies. However, if 5' untranslated and coding regions are omitted, normal ovarian expression is maintained but tissue specificity is relaxed: expression of the reporter gene is detected both in the ovary and in specific non-ovarian tissues of transformed females and males. The evidence suggests that the missing 5' untranslated and coding sequences may include negative elements that normally suppress expression in non-ovarian tissues, and that these putative elements are distinct from those that prevent premature expression in the ovarian follicles. The exact location of ectopic lacZ expression within the internal male genitalia depends on the constellation of 5' flanking chorion regulatory sequences included in the P element constructs. Ectopic expression of the CAT gene in the male genitalia under s15 promoter control can be abolished by mutating the hexamer TCACGT, a sequence previously shown to be essential for the normal expression of this chorion gene in the ovary.

Animals↗
PubMedDate not supplied

Primary B-cell response to neuropeptide Y and bovine pancreatic polypeptide.

An analysis of the murine primary response to protein epitopes has been made with two small highly structured proteins, neuropeptide Y (NPY) and bovine pancreatic polypeptide (BPP), both of 36-amino acid residue length and containing helical structures. A group of cell lines producing monoclonal IgM antibody have been prepared consisting of six anti-NPY and two anti-BPP. The VH nucleotide sequences have been determined and characterized as germ-line either by identity to established germ-line sequences or by inference from the germ-line character of the D and JH segments. The intrinsic association constants for the homologous ligands have been estimated to range from 10(4) to 10(7) M-1 based on competitive ELISA. No severe restriction in the utilization of VH families, D segments or JH segments appears to be involved in this response. Among the eight cell lines, three VH families were represented as well as all three families of D segments and all of the JH segments, although some preference for JH3 was indicated. The length of the N(D)N sequences was also not subject to restriction, ranging from 9 to 29. Two unusual features of the CRD3s were noted, one involving the utilization of an uncommon DSP2 segment and the other the apparent occurrence of a D-D fusion.

Amino Acid Sequence↗
PubMed1979

Demonstration of low density lipoprotein receptors in mouse teratocarcinoma stem cells and description of a method for producing receptor-deficient mutant mice.

Familial hypercholesterolemia, a widespread human genetic disorder implicated in vascular and coronary disease, has had no laboratory animal counterpart that would enable the pathogenesis to be analyzed and drugs to be tested in vivo. The primary lesion in some patients is known to occur in the cells' initial handling of the major cholesterol-carrying lipoprotein of plasma. It entails a deficiency in the specific cell surface receptor that binds low density lipoprotein (LDL), with a consequent alteration in the control of cholesterol metabolism. The present study was undertaken to devise a practical scheme for producing, from developmentally versatile mouse teratocarcinoma stem cells, whole-animal models with a comparable genetic lesion. This requires first learning whether the tumor stem cells in culture express LDL receptors, and next establishing a selection or screening procedure to identify receptor-deficient mutants in mutagenized cell cultures. The results show that the teratocarcinoma cells do in fact have specific high-affinity LDL receptors which are similar to those reported for fibroblasts and the parenchymal cells of specialized tissues and different from those of phagocytic cells. Sterols suppressed the otherwise efficient binding, internalization, and degradation of LDL ((125)I-labeled) by the cells. Acetylation of LDL blocked the binding. Only LDL and not high density lipoprotein (HDL) was bound. After LDL uptake and degradation, the liberated cholesterol led, as expected, to increased cholesteryl ester formation; it also suppressed activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase [HMG CoA reductase; mevalonate: NADP(+) oxidoreductase (CoA-acylating), EC 1.1.1.34], the rate-limiting step in cholesterol biosynthesis. Cells with LDL receptors were readily visualized by administering a fluorescent derivative of LDL; in the fluorescence microscope, labeling was seen in all cells. Cells with experimentally depressed receptors, yielding little fluorescence, were separable from those with normal fluorescence in the fluorescence-activated cell sorter. Thus, two methods for isolating receptor-deficient cells from mutagenized cultures are now available, either by visual recognition of low-fluorescing or nonfluorescing colonies in culture plates or by electronic cell sorting. Such mutants in an appropriate line of teratocarcinoma cells can then be passaged into blastocysts for full somatic tissue differentiation and germ-line development into mice.

Animals↗
PubMed1992

Selective disruption of genes expressed in totipotent embryonal stem cells.

Two retrovirus promoter trap vectors (U3His and U3Neo) have been used to disrupt genes expressed in totipotent murine embryonal stem (ES) cells. Selection in L-histidinol or G418 produced clones in which the coding sequences for histidinol-dehydrogenase or neomycin-phosphotransferase were fused to sequences in or near the 5' exons of expressed genes, including one in the developmentally regulated REX-1 gene. Five of seven histidinol-resistant clones and three of three G418-resistant clones generated germ-line chimeras. A total of four disrupted genes have been passed to the germ line, of which two resulted in embryonic lethalities when bred to homozygosity. The ability to screen large numbers of recombinant ES cell clones for significant mutations, both in vitro and in vivo, circumvents genetic limitations imposed by the size and long generation time of mice and will facilitate a functional analysis of the mouse genome.

Alcohol Oxidoreductases↗