Generation of mice with a 200-kb amyloid precursor protein gene deletion by Cre recombinase-mediated site-specific recombination in embryonic stem cells.

Gene disruptions and deletions of up to 20kb have been generated by homologous recombination with appropriate targeting vectors in murine embryonic stem (ES) cells. Because we could not obtain a deletion of about 200 kb in the mouse amyloid precursor protein gene by the classical technique, we employed strategies involving the insertion of loxP sites upstream and downstream of the region to be deleted by homologous recombination and elicited excision of the loxP-flanked region by introduction of a Cre expression vector into the ES cells. In the first approach, the loxP sequences were inserted in two successive steps and after each step, ES cell clones were isolated and characterized. Deletion of the loxP-flanked sequence was accomplished by introducing the cre gene in a third step. In the second approach, ES cells containing the upstream loxP cassette were electroporated simultaneously with the downstream loxP targeting vector and the Cre expression plasmid. ES cells were obtained that gave rise to chimeric mice capable of germ-line transmission of the deleted amyloid precursor protein allele.

Human semaphorins A(V) and IV reside in the 3p21.3 small cell lung cancer deletion region and demonstrate distinct expression patterns.

Semaphorins and collapsins make up a family of conserved genes that encode nerve growth cone guidance signals. We have identified two additional members of the human semaphorin family [human semaphorin A(V) and human semaphorin IV] in chromosome region 3p21.3, where several small cell lung cancer (SCLC) cell lines exhibit homozygous deletions indicative of a tumor suppressor gene. Human semaphorin A(V) has 86% amino acid homology with murine semaphorin A, whereas semaphorin IV is most closely related to murine semaphorin E, with 50% homology. These semaphorin genes are approximately 70 kb apart flanking two GTP-binding protein genes, GNAI-2 and GNAT-1. In contrast, other human semaphorin gene sequences (human semaphorin III and homologues of murine semaphorins B and C) are not located on chromosome 3. Human semaphorin A(V) is translated in vitro into a 90-kDa protein, which accumulates at the endoplasmic reticulum. The human semaphorin A(V) (3.4-kb mRNA) and IV (3.9- and 2.9-kb mRNAs) genes are expressed abundantly but differentially in a variety of human neural and nonneural tissues. Human semaphorin A(V) was expressed in only 1 out of 23 SCLCs and 7 out of 16 non-SCLCs, whereas semaphorin IV was expressed in 19 out of 23 SCLCs and 13 out of 16 non-SCLCs. Mutational analysis in semaphorin A(V) revealed mutations (germ line in one case) in 3 of 40 lung cancers. Our data suggest the need to determine the function of human semaphorins A(V) and IV in nonneural tissues and their role in the pathogenesis of lung cancer.

Group B streptococci escape host immunity by deletion of tandem repeat elements of the alpha C protein.

Group B streptococci (GBS) are the most common cause of neonatal sepsis, pneumonia, and meningitis. The alpha C protein is a surface-associated antigen; the gene (bca) for this protein contains a series of tandem repeats (each encoding 82 aa) that are identical at the nucleotide level and express a protective epitope. We previously reported that GBS isolates from two of 14 human maternal and neonatal pairs differed in the number of repeats contained in their alpha C protein; in both pairs, the alpha C protein of the neonatal isolate was smaller in molecular size. We now demonstrate by PCR that the neonatal isolates contain fewer tandem repeats. Maternal isolates were susceptible to opsonophagocytic killing in the presence of alpha C protein-specific antiserum, whereas the discrepant neonatal isolates proliferated. An animal model was developed to further study this phenomenon. Adult mice passively immunized with antiserum to the alpha C protein were challenged with an alpha C protein-expressing strain of GBS. Splenic isolates of GBS from these mice showed a high frequency of mutation in bca--most commonly a decrease in repeat number. Isolates from non-immune mice were not altered. Spontaneous deletions in the repeat region were observed at a much lower frequency (6 x 10(-4)); thus, deletions in that region are selected for under specific antibody pressure and appear to lower the organism's susceptibility to killing by antibody specific to the alpha C protein. This mechanism of antigenic variation may provide a means whereby GBS evade host immunity.

T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination.

UDP-N-acetylgalactosamine (GalNAc): polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc-T) catalyzes transfer of the monosaccharide GalNAc to serine and threonine residues, thereby initiating O-linked oligosaccharide biosynthesis. Previous studies have suggested the possibility of multiple polypeptide GalNAc-Ts, although attachment of saccharide units to polypeptide or lipid in generating oligosaccharide structures in vertebrates has been dependent upon the activity of single gene products. To address this issue and to determine the relevance of Oglycosylation variation in T-cell ontogeny, we have directed Cre/loxP mutagenic recombination to the polypeptide GalNAc-T locus in gene-targeted mice. Resulting deletion in the catalytic region of polypeptide GalNAc-T occurred to completion on both alleles in thymocytes and was found in peripheral T cells, but not among other cell types. Thymocyte O-linked oligosaccharide formation persisted in the absence of a functional targeted polypeptide GalNAc-T allele as determined by O-glycan-specific lectin binding. T-cell development and colonization of secondary lymphoid organs were also normal. These results indicate a complexity in vertebrate O-glycan biosynthesis that involves multiple polypeptide GalNAc-Ts. We infer the potential for protein-specific O-glycan formation governed by distinct polypeptide GalNAc-Ts.

Y chromosome short arm-Sxr recombination in XSxr/Y males causes deletion of Rbm and XY female sex reversal.

We earlier described three lines of sex-reversed XY female mice deleted for sequences believed close to the testes-determining gene (Sry) on the Y chromosome short arm (Yp). The original sex-reversed females appeared among the offspring of XY males that carried the Yp duplication Sxr on their X chromosome. Earlier cytogenetic observations had suggested that the deletions resulted from asymmetrical meiotic recombination between the Y and the homologous Sxr region, but no direct evidence for this hypothesis was available. We have now analyzed the offspring of XSxr/Y males carrying an evolutionarily divergent Mus musculus domesticus Y chromosome, which permits detection and characterization of such recombination events. This analysis has enabled the derivation of a recombination map of Yp and Sxr, also demonstrating the orientation of Yp with respect to the Y centromere. The mapping data have established that Rbm, the murine homologue of a gene family cloned from the human Y chromosome, lies between Sry and the centromere. Analysis of two additional XY female lines shows that asymmetrical Yp-Sxr recombination leading to XY female sex reversal results in deletion of Rbm sequences. The deletions bring Sry closer to Y centromere, consistent with the hypothesis that position-effect inactivation of Sry is the basis for the sex reversal.

Deletion of high-avidity T cells by thymic epithelium.

Tolerance induction by thymic epithelium induces a state of so-called "split tolerance," characterized in vivo by tolerance and in vitro by reactivity to a given thymically expressed antigen. Using a model major histocompatibility complex class I antigen, H-2Kb (Kb), three mechanisms of thymic epithelium-induced tolerance were tested: induction of tolerance of tissue-specific antigens exclusively, selective inactivation of T helper cell-independent cytotoxic T lymphocytes, and deletion of high-avidity T cells. To this end, thymic anlagen from Kb-transgenic embryonic day 10 mouse embryos, taken before colonization by cells of hemopoietic origin, were grafted to nude mice. Tolerance by thymic epithelium was not tissue-specific, since Kb-bearing skin and spleen grafts were maintained indefinitely. Only strong priming in vivo could partially overcome the tolerant state and induce rejection of some skin grafts overexpressing transgenic Kb. Furthermore, the hypothesis that thymic epithelium selectively inactivates those T cells that reject skin grafts in a T helper-independent fashion could not be supported. Thus, when T-cell help was provided by a second skin graft bearing an additional major histocompatibility complex class II disparity, tolerance to the Kb skin graft was not broken. Finally, direct evidence could be obtained for the avidity model of thymic epithelium-induced negative selection, using Kb-specific T-cell receptor (TCR) transgenic mice. Thymic epithelium-grafted TCR transgenic mice showed a selective deletion of those CD8+ T cells with the highest density of the clonotypic TCR. These cells presumably represent the T cells with the highest avidity for Kb. We conclude that split tolerance induced by thymic epithelium was mediated by the deletion of those CD8+ T lymphocytes that have the highest avidity for antigen.

Mouse model of human beta zero thalassemia: targeted deletion of the mouse beta maj- and beta min-globin genes in embryonic stem cells.

beta zero-Thalassemia is an inherited disorder characterized by the absence of beta-globin polypeptides derived from the affected allele. The molecular basis for this deficiency is a mutation of the adult beta-globin structural gene or cis regulatory elements that control beta-globin gene expression. A mouse model of this disease would enable the testing of therapeutic regimens designed to correct the defect. Here we report a 16-kb deletion that includes both adult beta-like globin genes, beta maj and beta min, in mouse embryonic stem cells. Heterozygous animals derived from the targeted cells are severely anemic with dramatically reduced hemoglobin levels, abnormal red cell morphology, splenomegaly, and markedly increased reticulocyte counts. Homozygous animals die in utero; however, heterozygous mice are fertile and transmit the deleted allele to progeny. The anemic phenotype is completely rescued in progeny derived from mating beta zero-thalassemic animals with transgenic mice expressing high levels of human hemoglobin A. The beta zero-thalassemic mice can be used to test genetic therapies for beta zero-thalassemia and can be bred with transgenic mice expressing high levels of human hemoglobin HbS to produce an improved mouse model of sickle cell disease.

Pleiotropy in microdeletion syndromes: neurologic and spermatogenic abnormalities in mice homozygous for the p6H deletion are likely due to dysfunction of a single gene.

Variability and complexity of phenotypes observed in microdeletion syndromes can be due to deletion of a single gene whose product participates in several aspects of development or can be due to the deletion of a number of tightly linked genes, each adding its own effect to the syndrome. The p6H deletion in mouse chromosome 7 presents a good model with which to address this question of multigene vs. single-gene pleiotropy. Mice homozygous for the p6H deletion are diluted in pigmentation, are smaller than their littermates, and manifest a nervous jerky-gait phenotype. Male homozygotes are sterile and exhibit profound abnormalities in spermiogenesis. By using N-ethyl-N-nitrosourea (EtNU) mutagenesis and a breeding protocol designed to recover recessive mutations expressed hemizygously opposite a large p-locus deletion, we have generated three noncomplementing mutations that map to the p6H deletion. Each of these EtNU-induced mutations has adverse effects on the size, nervous behavior, and progression of spermiogenesis that characterize p6H deletion homozygotes. Because EtNU is thought to induce primarily intragenic (point) mutations in mouse stem-cell spermatogonia, we propose that the trio of phenotypes (runtiness, nervous jerky gait, and male sterility) expressed in p6H deletion homozygotes is the result of deletion of a single highly pleiotropic gene. We also predict that a homologous single locus, quite possibly tightly linked and distal to the D15S12 (P) locus in human chromosome 15q11-q13, may be associated with similar developmental abnormalities in humans.

Identification by representational difference analysis of a homozygous deletion in pancreatic carcinoma that lies within the BRCA2 region.

Homozygous deletions have been central to the discovery of several tumor-suppressor genes, but their finding has often been either serendipitous or the result of a directed search. A recently described technique [Lisitsyn, N., Lisitsyn, N. & Wigler, M. (1993) Science 259, 946-951] held out the potential to efficiently discover such events in an unbiased manner. Here we present the application of the representational difference analysis (RDA) to the study of cancer. We cloned two DNA fragments that identified a homozygous deletion in a human pancreatic adenocarcinoma, mapping to a 1-centimorgan region at chromosome 13q12.3 flanked by the markers D13S171 and D13S260. Interestingly, this lies within the 6-centimorgan region recently identified as the BRCA2 locus of heritable breast cancer susceptibility. This suggests that the same gene may be involved in multiple tumor types and that its function is that of a tumor suppressor rather than that of a dominant oncogene.

A region of consistent deletion in neuroblastoma maps within human chromosome 1p36.2-36.3.

Deletion of the short arm of human chromosome 1 is the most common cytogenetic abnormality observed in neuroblastoma. To characterize the region of consistent deletion, we performed loss of heterozygosity (LOH) studies on 122 neuroblastoma tumor samples with 30 distal chromosome 1p polymorphisms. LOH was detected in 32 of the 122 tumors (26%). A single region of LOH, marked distally by D1Z2 and proximally by D1S228, was detected in all tumors demonstrating loss. Also, cells from a patient with a constitutional deletion of 1p36, and from a neuroblastoma cell line with a small 1p36 deletion, were analyzed by fluorescence in situ hybridization. Cells from both sources had interstitial deletions of 1p36.2-36.3 which overlapped the consensus region of LOH defined by the tumors. Interstitial deletion in the constitutional case was confirmed by allelic loss studies using the panel of polymorphic markers. Four proposed candidate genes--DAN, ID3 (heir-1), CDC2L1 (p58), and TNFR2--were shown to lie outside of the consensus region of allelic loss, as defined by the above deletions. These results more precisely define the location of a neuroblastoma suppressor gene within 1p36.2-36.3, eliminating 33 centimorgans of proximal 1p36 from consideration. Furthermore, a consensus region of loss, which excludes the four leading candidate genes, was found in all tumors with 1p36 LOH.

Differential subcellular mRNA targeting: deletion of a single nucleotide prevents the transport to axons but not to dendrites of rat hypothalamic magnocellular neurons.

It has previously been shown that mRNA encoding the arginine vasopressin (AVP) precursor is targeted to axons of rat magnocellular neurons of the hypothalamo-neurohypophyseal tract. In the homozygous Brattle-boro rat, which has a G nucleotide deletion in the coding region of the AVP gene, no such targeting is observed although the gene is transcribed. RNase protection and heteroduplex analyses demonstrate that, in heterozygous animals, which express both alleles of the AVP gene, the wild-type but not the mutant transcript is subject to axonal compartmentation. In contrast, wild-type and mutant AVP mRNAs are present in dendrites. These data suggest the existence of different mechanisms for mRNA targeting to the two subcellular compartments. Axonal mRNA localization appears to take place after protein synthesis; the mutant transcript is not available for axonal targeting because it lacks a stop codon preventing its release from ribosomes. Dendritic compartmentation, on the other hand, is likely to precede translation and, thus, would be unable to discriminate between the two mRNAs.

Procedures for completion and deletion of national priorities list sites.

Mode of access: Internet.

Stem-completion priming for words studied in sentences: The context deletion effect under direct and indirect memory instructions

Two studies investigated the context deletion effect, the attenuation of priming in implicit memory tests of words when words have been studied in text rather than in isolation. In Experiment 1, stem completion for single words was primed to a greater extent by words studied alone than in sentence contexts, and a higher proportion of completions from studied words was produced under direct instructions (cued recall) than under indirect instructions (produce the first completion that comes to mind). The effect of a sentence context was eliminated when participants were instructed to attend to the target word during the imagery generation task used in the study phase. In Experiment 2, the effect of a sentence context at study was reduced when the target word was presented in distinctive format within the sentence, and the study task (grammatical judgment) was directed at a word other than the target. The results implicate conceptual and perceptual processes that distinguish a word from its context in priming in word stem completion.