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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↗

PubMed1991

More is not better: brood size and population growth in a self-fertilizing nematode.

The normal form of the nematode Caenorhabditis elegans is a self-fertilizing hermaphrodite, which produces from the same germ-line tissue first a limited number of sperm and then a larger number of oocytes. Self-progeny brood sizes are determined by the number of sperm, and most of the oocytes remain unfertilized. Therefore it might seem selectively advantageous to increase the number of sperm, and hence the size of the brood. A mutation that leads to a 50% increase in sperm production allows a comparison of population growth rates between the wild type (mean brood 327 progeny) and the mutant (mean brood 499 progeny). Wild-type populations grow faster, as measured by food consumption, indicating that increased brood size is not advantageous. The mutant appears to be at a disadvantage because the additional spermatogenesis leads to a delay in the onset of oogenesis, and hence to an increase in the minimum generation time. In support of the notion of an optimal brood size, it was found that different natural isolates of this species have self-fertilities similar to that of the standard laboratory strain, in the range 250-350 progeny per worm.

Animals↗

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↗

PubMed1991

Carboxy-terminal truncation activates glp-1 protein to specify vulval fates in Caenorhabditis elegans.

The glp-1 and lin-12 genes encode homologous transmembrane proteins that may act as receptors for cell interactions during development. The glp-1 product is required for induction of germ-line proliferation and for embryogenesis. By contrast, lin-12 mediates somatic cell interactions, including those between the precursor cells that form the vulval hypodermis (VPCs). Here we analyse an unusual allele of glp-1, glp-1(q35), which displays a semidominant multivulva phenotype (Muv), as well as the typical recessive, loss-of-function Glp phenotypes (sterility and embryonic lethality). We find that the effects of glp-1(q35) on VPC development mimic those of dominant lin-12 mutations, even in the absence of lin-12 activity. The glp-1(q35) gene bears a nonsense mutation predicted to eliminate the 122 C-terminal amino acids, including a ProGluSerThr (PEST) sequence thought to destabilize proteins. We suggest that the carboxy terminus bears a negative regulatory domain which normally inactivates glp-1 in the VPCs. We propose that inappropriate glp-1(q35) activity can substitute for lin-12 to determine vulval fate, perhaps by driving the VPCs to proliferate.

Amino Acid Sequence↗

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↗

PubMed1991

Molecular characterization of a supratypic cross-reactive idiotype associated with IgM autoantibodies.

Neoplastic B cells from patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphomas (SLL) frequently express surface Ig reactive with the mouse mAb, Lc1. Raised against a human monoclonal IgM with rheumatoid factor activity, Lc1 detects a major cross-reactive Id (CRI) present on the H chain of many monoclonal IgM autoantibodies. In contrast to other major autoantibody-CRI investigated to date, we note that the Lc1-CRI is expressed by subpopulation of cells in the germinal centers, as well as in the mantle zones, of secondary human B cell follicles. To examine the molecular basis for Lc1 expression, we used the polymerase chain reaction to isolate the functionally rearranged Ig VH genes of monoclonal Lc1-reactive B cell populations from six unrelated patients with CLL or SLL. Although the neoplastic B cells from most patients with CLL or SLL express the CD5 surface differentiation Ag, the lymphoma cells from one patient with SLL were CD5-negative. We find that the Lc1-reactive cells from each cell population have Ig rearrangements involving a VH gene of the VH4 subgroup. However, the VH4 genes rearranged in different Lc1-reactive tumor populations may originate from at least two disparate germ-line VH4 genes. Also, in contrast to the CD5-positive tumor populations, we find evidence for intraclonal diversity in the functionally rearranged VH4 genes of the CD5-negative SLL. Collectively, this study discerns a degeneracy in the VH4 genes that can encode the Lc1 CRI, indicating the term "supratypic cross-reactive idiotype" may best describe the specificity of the Lc1 mAb. Also, this study suggests that expression of CD5 may delineate categories of B cell SLL that differ in their relative rates of constitutive Ig V gene somatic mutation.

Amino Acid Sequence↗

PubMed1992

Sequencing heavy- and light-chain variable genes of single B-hybridoma cells by total enzymatic amplification.

We have devised a protocol to obtain accurate and complete sequences of the immunoglobulin heavy- and light-chain variable-region (VH and VL) genes of single B-hybridoma cells that express defined V genes. The amplification achieved ranges from 2 x 10(13)- to 1 x 10(14)-fold. Only one potential Taq DNA polymerase error was observed in 7590 nucleotides of sequence, thus permitting the identification of naturally occurring somatic mutations. The two-step nature of the amplification protocol provides sufficient DNA for a minimum of 160 sets of sequencing reactions of both the VH and VL genes from one cell without cloning. The amplification of relatively long segments of DNA in the first step of the protocol permits second-step amplification and sequencing of regions that flank VH and VL codons. Fractionating cellular lysates prior to the first step of amplification permits the separate amplification of V genes on opposite sister chromatids and possibly on opposite strands of the same DNA duplex. Accurate sequencing of VH and VL genes of defined germ-line origin that are expressed by single B cells taken directly from the animal is thus made feasible by this approach.

Animals↗

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↗

PubMed1992

Intracellular selection, conversion bias, and the expected substitution rate of organelle genes.

A key step in the substitution of a new organelle mutant throughout a population is the generation of germ-line cells homoplasmic for that mutant. Given that each cell typically contains multiple copies of organelles, each of which in turn contains multiple copies of the organelle genome, processes akin to drift and selection in a population are responsible for producing homoplasmic cells. This paper examines the expected substitution rate of new mutants by obtaining the probability that a new mutant is fixed throughout a cell, allowing for arbitrary rates of genome turnover within an organelle and organelle turnover within the cell, as well as (possibly biased) gene conversion and genetic differences in genome and/or organelle replication rates. Analysis is based on a variation of Moran's model for drift in a haploid population. One interesting result is that if the rate of unbiased conversion is sufficiently strong, it creates enough intracellular drift to overcome even strong differences in the replication rates of wild-type and mutant genomes. Thus, organelles with very high conversion rates are more resistant to intracellular selection based on differences in genome replication and/or degradation rates. It is found that the amount of genetic exchange between organelles within the cell greatly influences the probability of fixation. In the absence of exchange, biased gene conversion and/or differences in genome replication rates do not influence the probability of fixation beyond the initial fixation within a single organelle. With exchange, both these processes influence the probability of fixation throughout the entire cell. Generally speaking, exchange between organelles accentuates the effects of directional intracellular forces.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Replication↗

PubMed1991

V lambda and J lambda-C lambda gene segments of the human immunoglobulin lambda light chain locus are separated by 14 kb and rearrange by a deletion mechanism.

We have cloned a region of 124 kb of the human immunoglobulin lambda light chain locus on chromosome 22 encompassing seven V lambda and seven J-C lambda gene segments. No further C lambda gene segment was found in a region of 35 kb downstream of C lambda 7, which encodes the Ke+Oz- isotype. The C lambda proximal V lambda gene segment V lambda III. 1 is located 14.5 kb upstream of C lambda 1. The five sequenced V lambda genes have the same transcriptional orientation as the J-C lambda gene segments which is likely to be true for the majority of the V lambda gene segments in the human lambda locus and which suggests a deletion mechanism for DNA rearrangement. This is supported by hybridization of V lambda gene probes to germ-line and rearranged DNA from lambda light chain-producing cell lines. Sequences of 23 cDNA clones allow to establish a V lambda subgroup classification based on nucleic acid sequence data and an estimate of the J-C lambda usage.

Amino Acid Sequence↗

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↗

PubMed1992

Identification of a genomic DNA fragment containing the Drosophila melanogaster ovarian tumor gene (otu) and localization of regions governing its expression.

We have identified a genomic DNA fragment which restores fertility to mutants of the ovarian tumor locus (otu) of Drosophila melanogaster. Germ-line transformants bearing this fragment express otu mRNA with the same tissue specificity as, and at levels comparable to, the wild-type otu gene. Transcription from the otu promoter, P(otu), which lacks a TATA element, appears to be initiated at multiple transcription start points (tsp) within an 80-bp region. Deletion of sequences upstream of the tsp indicates that a region between nucleotides -190 and -310 is required for proper expression from the otu gene. A DNA fragment containing 452 bp upstream and 126 bp downstream from the tsp is able to direct expression of the Escherichia coli lacZ gene in the germ cells of the ovary and testis, indicating that cis-acting regulatory elements governing these expression patterns are located in a 578-bp region surrounding the multiple tsp.

Animals↗

PubMed1991

Effects of the steel gene product on mouse primordial germ cells in culture.

Mutations at the steel (sl) and dominant white spotting (W) loci in the mouse affect primordial germ cells (PGC), melanoblasts and haemopoietic stem cells. The W gene encodes a cell-surface receptor of the tyrosine kinase family, the proto-oncogene c-kit. In situ analysis has shown c-kit messenger RNA expression in PGC in the early genital ridges. The Sl gene encodes the ligand for this receptor, a peptide growth factor, called here stem cell factor (SCF). SCF mRNA is expressed in many regions of the early mouse embryo, including the areas of migration of these cell types. It is important now to identify the role of the Sl-W interaction in the development of these migratory embryonic stem cell populations. Using an in vitro assay system, we show that SCF increases both the overall numbers and colony sizes of migratory PGC isolated from wild-type mouse embryos, and cultured on irradiated feeder layers of STO cells (a mouse embryonic fibroblast line). In the absence of feeder cells, SCF causes a large increase in the initial survival and apparent motility of PGC in culture. But labelling with bromodeoxyuridine shows that SCF is not, by itself, a mitogen for PGC. SCF does not exert a chemotropic effect on PGC in in vitro assays. These results suggest that SCF in vivo is an essential requirement for PGC survival. This demonstrates the control of the early germ-line population by a specific trophic factor.

Animals↗

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↗