Reverse genetic studies of homologous DNA recombination using the chicken B-lymphocyte line, DT40

DT40 is an avian leucosis virus-transformed chicken B-lymphocyte line which exhibits high ratios of targeted to random integration of transfected DNA constructs. This efficient targeted integration may be related to the ongoing diversification of the variable segment of the immunoglobulin gene through homologous DNA recombination-controlled gene conversion. DT40s are a convenient model system for making gene-targeted mutants. Another advantage is the relative tractability of these cells, which makes it possible to disrupt multiple genes in a single cell and to generate conditionally gene-targeted mutants including temperature-sensitive mutants. There are strong phenotypic similarities between murine and DT40 mutants of various genes involved in DNA recombination. These similarities confirm that the DT40 cell line is a reasonable model for the analysis of vertebrate DNA recombination, despite obvious concerns associated with the use of a transformed cell line, which may have certain cell-line-specific characteristics. Here we describe our studies of homologous DNA recombination in vertebrate somatic cells using reverse genetics in DT40 cells.     

Direct cDNA cloning of the rearranged immunoglobulin variable region

A major problem in the study of multigene families is the effort required to clone and sequence these genes. We describe a method to rapidly clone and sequence immunoglobulin variable region gene sequences without constructing cDNA libraries. Because immunoglobulin variable-region genes are flanked by conserved sequences, we have been able to apply the polymerase chain reaction (PCR) to clone and sequence both the light- and heavy-chain rearranged immunoglobulin genes from small numbers of hybridoma cells. This method will greatly facilitate the construction of chimeric mouse/human monoclonal antibodies for immunoglobulin structural studies as well as for therapeutic use.     

Novartis Medal Lecture. Antibodies: a paradigm for the evolution of molecular recognition

Novel proteins have been elaborated over evolutionary time by an iterative alternation of mutation and selection. In a similar way, the humoral immune system also uses an iterative alternation of mutation and selection to generate novel antibodies that display a high affinity for their cognate antigen -- but this is achieved in a matter of a days. Gene rearrangement is used to produce a primary repertoire of antibodies and, on entering the body, antigen triggers the clonal expansion of those B lymphocytes that express a cognate antibody, albeit one of low affinity. Rapid and specific affinity maturation is then achieved by subjecting the immunoglobulin genes in the rapidly expanding B cells to a period of intense mutation. The intensity of this mutational assault is tolerated because it is targeted specifically to the immunoglobulin genes, causing relatively little damage to other loci. Antigen-mediated selection then allows the preferential expansion of those mutants expressing antibodies displaying improved binding characteristics. Here, studies are described that have been performed to glean insight into the mechanisms of the hypermutation and selection processes. Experiments are also described in which an attempt has been made to recapitulate aspects of physiological antibody generation in vitro, allowing the development of novel approaches to the generation of proteins with high-affinity binding sites.     

Immunoglobulin molecular genetics: the prospects for mutational analysis of the chromosomal immunoglobulin genes

Homologous recombination between transferred and chromosomal DNA can be used to effect precise, predetermined modifications of the chromosomal genes. Ultimately this phenomenon should allow the assessment of genetic regulatory elements as they function in the normal chromosomal environment. We have previously described a system for isolating mutant hybridoma cells that are defective in immunoglobulin (Ig) production, with a view toward using these mutants to define cis-acting elements that influence Ig gene expression. Here we describe results that indicate that homologous recombination between transferred and chromosomal Ig genes can be used to map Ig mutations by marker rescue.     

Comparative analyses of immunoglobulin genes: surprises and portents

The study of immunoglobulin genes in non-mouse and non-human models has shown that different vertebrate groups have evolved distinct methods of generating antibody diversity. By contrast, the development of T cells in the thymus is quite similar in all of the species that have been examined. The three mechanisms by which B cells uniquely modify their immunoglobulin genes -- somatic hypermutation, gene conversion and class switching -- are increasingly believed to share some fundamental mechanisms, which studies in different vertebrate groups have helped (and will continue to help) to resolve. When these mechanisms are better understood, we should be able to look to the constitutive pathways from which they have evolved and perhaps determine whether the rearrangement of variable, diversity and joining antibody gene segments -- V(D)J recombination -- was superimposed on an existing adaptive immune system.     

Genetic analysis of early endocrine pancreas formation in zebrafish

Endocrine pancreas of zebrafish consist of at least four different cell types that function similarly to mammalian pancreatic islet. No mutants specifically affecting formation of the endocrine pancreas have been identified during the previous large-scale mutagenesis screens in zebrafish due to invisibility of a pancreatic islet. We combined in situ hybridization method to visualize pancreatic islet with an ethyl-nitroso-urea mutagenesis screen to identify novel genes involved in pancreatic islet formation in zebrafish. We screened 900 genomes and identified 11 mutations belonging to nine different complementation groups. These mutants fall into three major phenotypic classes displaying severely reduced insulin expression, reduced insulin expression with abnormal islet morphology, or abnormal islet morphology with relatively normal number of insulin expressing cells. Seven of these mutants do not have any other visible phenotypes associated. These mutations affect different processes in pancreatic islet development. Additional analysis on glucagon and somatostatin cell specification revealed that somatostatin cells are specified at a separate domain from insulin cells whereas glucagon cells are specified adjacent to insulin cells. Furthermore, glucagon cells and somatostatin cells are always associated with insulin cells in mutants that have scattered insulin expression. These data indicate that there are separate mechanisms regulating endocrine cell migration, proliferation, and differentiation. Further study on these mutants will reveal important information on novel genes involved in pancreatic islet cell specification and morphogenesis.     

Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea

Immunoglobulin heavy chain genes from Raja erinacea have been isolated by cross hybridization with probes derived from the immunoglobulin genes of Heterodontus francisci (horned shark), a representative of a different elasmobranch order. Heavy chain variable (VH), diversity (DH) and joining (JH) segments are linked closely to constant region (CH) exons, as has been described in another elasmobranch. The nucleotide sequence homology of VH gene segments within Raja and between different elasmobranch species is high, suggesting that members of this phylogenetic subclass may share one VH family. The organization of immunoglobulin genes segments is diverse; both VD-J and VD-DJ joined genes have been detected in the genome of non-lymphoid cells. JH segment sequence diversity is high, in contrast to that seen in a related elasmobranch. These data suggest that the clustered V-D-J-C form of immunoglobulin heavy chain organization, including germline joined components, may occur in all subclasses of elasmobranchs. While variation in VH gene structure is limited, gene organization appears to be diverse.     

Effects of anoxia and the mitochondrion on expression of aerobic nuclear COX genes in yeast: evidence for a signaling pathway from the mitochondrial genome to the nucleus

Eucaryotic cells contain at least two general classes of oxygen-regulated nuclear genes: aerobic genes and hypoxic genes. Hypoxic genes are induced upon exposure to anoxia while aerobic genes are down-regulated. Recently, it has been reported that induction of some hypoxic nuclear genes in mammals and yeast requires mitochondrial respiration and that cytochrome-c oxidase functions as an oxygen sensor during this process. In this study, we have examined the role of the mitochondrion and cytochrome-c oxidase in the expression of yeast aerobic nuclear COX genes. We have found that the down-regulation of these genes in anoxic cells is reflected in reduced levels of their subunit polypeptides and that cytochrome-c oxidase subunits I, II, III, Vb, VI, VII, and VIIa are present in promitochondria from anoxic cells. By using nuclear cox mutants and mitochondrial rho(0) and mit(-) mutants, we have found that neither respiration nor cytochrome-c oxidase is required for the down-regulation of these genes in cells exposed to anoxia but that a mitochondrial genome is required for their full expression under both normoxic and anoxic conditions. This requirement for a mitochondrial genome is unrelated to the presence or absence of a functional holocytochrome-c oxidase. We have also found that the down-regulation of these genes in cells exposed to anoxia and the down-regulation that results from the absence of a mitochondrial genome are independent of one another. These findings indicate that the mitochondrial genome, acting independently of respiration and oxidative phosphorylation, affects the expression of the aerobic nuclear COX genes and suggest the existence of a signaling pathway from the mitochondrial genome to the nucleus.     

Prevacuolar compartment morphology in vps mutants of Saccharomyces cerevisiae

Over 60 genes have been identified that affect protein sorting to the lysosome-like vacuole in Saccharomyces cerevisiae. Cells with mutations in these vacuolar protein sorting (vps) genes fall into seven general classes based upon their vacuolar morphology. Class A mutants have a morphologically wild type vacuole, while Class B mutants have a fragmented vacuole. There is no discernable vacuolar structure in Class C mutants. Class D mutants have a slightly enlarged vacuole, but Class E mutants have a normal looking vacuole with an enlarged prevacuolar compartment (PVC), which is analogous to the mammalian late endosome. Class F mutants have a wild type appearing vacuole as well as fragmented vacuolar structures. vps mutants have also been found with a tubulo-vesicular vacuole structure. vps mutant morphology is pertinent, as mutants of the same class may work together and/or have a block in the same general step in the vacuolar protein sorting pathway. We probed PVC morphology and location microscopically in live cells of several null vps mutants using a GFP fusion protein of Nhx1p, an Na(+)/H(+) exchanger normally localized to the PVC. We show that cell strains deleted for VPS proteins that have been previously shown to work together, regardless of VPS Class, have the same PVC morphology. Cell strains lacking VPS genes that have not been implicated in the same pathway show different PVC morphologies, even if the mutant strains are in the same VPS Class. These new studies indicate that PVC morphology is another tier of classification that may more accurately identify proteins that function together in vacuolar protein sorting than the original vps mutation classes.     

Cell-type preference of immunoglobulin kappa and lambda gene promoters.

Immunoglobulin gene constant regions are known to be associated with strictly tissue-specific enhancer elements. Until recently the promoter of the variable region, which becomes linked to the constant region by somatic rearrangement, could have been viewed as a passive recipient of the enhancer stimulus. Here we show that the promoters of the immunoglobulin kappa and lambda light chain genes are approximately 20-30 times more active in lymphoid cells than in non-lymphoid cells. To avoid the problem of differential mRNA stability upon transfection of immunoglobulin genes into non-lymphoid cells we have constructed chimeric genes. All kappa mRNA sequences were progressively deleted to fuse the kappa gene promoter to a globin gene coding body. A similar chimeric gene was constructed with the promoter of the lambda gene. The cell-type preference of the promoter may be exploited during B-lymphocyte differentiation to regulate the immunoglobulin gene promoter independently from the enhancer.     

Plant mutant tubulin genes as marker selective genes for genetic engineering

The approaches for new marker genes usage in selection of transformed plant cells, which are based on using mutant tubulin genes from natural plant biotypes and, in perspective, from induced plant mutants have been considered. The results of investigations of plant (biotypes, mutants) resistance to herbicides with antimicrotubular mode of action on molecular and cellular levels have been summarized. The reports dealing with study the transferring and the expression of mutant tubulin genes conferring resistance to amiprophosmethyl (phosphorothioamidate herbicide) and to trifluralin (dinitroaniline herbicide) from corresponding N. plumbaginifolia mutants into related and remote plant species by somatic hybridisation methods have been analyzed. The results of experiments on monocotyledonous and dicotyledonous. plant transformation by mutant alpha-tubulin gene conferring resistance to dinitroanilines are described to test the possibility of its using as a marker gene with obtaining, at the same time, a dinitroaniline-resistant plants.     

Genetic analysis of cell morphogenesis in fission yeast--a role for casein kinase II in the establishment of polarized growth.

We have initiated a study to identify genes regulating cell morphogenesis in the fission yeast Schizosaccharomyces pombe. Five genes have been identified, orb1-orb5, whose mutation gives rise to spherical cells, indicative of an inability to polarize growth. Two further genes have been identified, tea1 and ban1, whose mutant alleles have disturbed patterns of tip growth, leading to T-shaped and curved cells. In fission yeast, sites of cell wall deposition are defined by actin localization, with actin distributions and therefore growth patterns undergoing cell cycle stage-specific reorganization. Studies of double mutants constructed between orb5-19 and various cdc mutants blocked before and after cell division show that orb5 is required for the re-establishment of polar growth following cytokinesis. This indicates that the mutant allele orb5-19 is defective in the reinitiation of polarized growth, even though actin reorganization to the cell tips occurs normally. orb5 encodes a fission yeast homologue of casein kinase II alpha. We propose that this kinase plays a role in the translation of cell polarity into polarized growth, but not in the establishment of polarity itself.     

Complementation and the physiological characteristics of the ts mutants of Pseudomonas aeruginosa phage SM

Seventy three temperature-sensitive mutants of Pseudomonas aeruginosa SM phage have been obtained using different mutagens and assigned to thirteen complementation groups. Representative mutants of each group have been studied with the aim of characterizing tentatively the time of genes expression in infected bacteria. Two genes appear to function during the first minutes after infection, whereas the remaining genes are needed for late functions. Most of the temperature-sensitive functions in the different mutants are reversible, i.e. they become active when the infected cells are shifted-down to the permissive temperature.     

Temperature-Sensitive Nonsense Mutations in Essential Genes of Escherichia coli

Cells containing nonsense mutations in essential genes have been isolated in a strain of Escherichia coli that carried the su4(ts) gene which specifies a temperature-sensitive tyrosine transfer ribonucleic acid. Such cells are unable to form colonies at temperatures which inactivate this suppressor transfer ribonucleic acid. A screening procedure for the identification of mutants that carry temperature-sensitive nonsense mutations in essential genes is described, and certain properties of two such mutants are reported.     

Many human immunoglobulin heavy-chain IGHV gene polymorphisms have been reported in error

The identification of the genes that make up rearranged immunoglobulin genes is critical to many studies. For example, the enumeration of mutations in immunoglobulin genes is important for the prognosis of chronic lymphocytic leukemia, and this requires the accurate identification of the germline genes from which a particular sequence is derived. The immunoglobulin heavy-chain variable (IGHV) gene repertoire is generally considered to be highly polymorphic. In this report, we describe a bioinformatic analysis of germline and rearranged immunoglobulin gene sequences which casts doubt on the existence of a substantial proportion of reported germline polymorphisms. We report a five-level classification system for IGHV genes, which indicates the likelihood that the genes have been reported accurately. The classification scheme also reflects the likelihood that germline genes could be incorrectly identified in mutated VDJ rearrangements, because of similarities to other alleles. Of the 226 IGHV alleles that have previously been reported, our analysis suggests that 104 of these alleles almost certainly include sequence errors, and should be removed from the available repertoire. The analysis also highlights the presence of common mismatches, with respect to the germline, in many rearranged heavy-chain sequences, suggesting the existence of twelve previously unreported alleles. Sequencing of IGHV genes from six individuals in this study confirmed the existence of three of these alleles, which we designate IGHV3-49*04, IGHV3-49*05 and IGHV4-39*07. We therefore present a revised repertoire of expressed IGHV genes, which should substantially improve the accuracy of immunoglobulin gene analysis.     

A gene trap approach to identify genes that control development

One methodology called gene trap represents a versatile strategy by which murine genes that control developmental events can be captured and identified with corresponding mutants produced at the same time. Gene trap methodology has been developed and several genes and their mutants have been analyzed, but almost all of the genes reported are those already known or murine homologs of other species. In this study, the efficiency of the gene trap methodology was improved and a novel mutant mouse strain named jumonji established which displayed an intriguing defect. Homozygous fetal mice died in utero and a significant proportion of the homozygotes showed abnormal groove formation on the neural plate and a defect in neural tube closure with a mixed genetic background of 129/Ola and BALB/c. The trapped gene believed to be responsible for these phenotypes encodes a novel nuclear protein. The results reveal that the gene trap approach can identify unknown interesting genes in murine development. The gene trap strategy, however, has several problems, the greatest of which is the difficulty in prescreening embryonic stem (ES) cells for interesting trapped genes. Recent studies are solving this problem and show that the prescreening of ES cells for genes with several characteristics is possible.