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P Leder

Publications and source records attributed to P Leder.

At least 127 records · Page 7Linked to original sources
PubMed1991

Parental-specific methylation of an imprinted transgene is established during gametogenesis and progressively changes during embryogenesis.

Genomic imprinting is a regulatory process that requires a cell to recognize the parental origin of alleles. To understand how these alleles are distinguished, we have assessed changes in the DNA methylation of an imprinted transgene as it switches from one inheritance pattern to another while moving through gametogenesis and embryogenesis. We find that both maternally and paternally inherited methylation patterns are erased in primordial germ cells and that distinctive patterns emerge during germ cell maturation. In the case of the maternal allele, the methylation pattern is fully acquired during oogenesis. In the case of the paternal allele, the methylation pattern found in sperm undergoes further modification during embryogenesis. Thus, the distinction between "erased" maternal and paternal alleles is first established during their residence in different germ cells and then may be maintained by the recognition of the distinctive patterns that each allele displays in the zygote.

Alleles↗
PubMed1991

Cytoplasmic tail deletion converts membrane immunoglobulin to a phosphatidylinositol-linked form lacking signaling and efficient antigen internalization functions.

Membrane-bound immunoglobulin (mIg) is the antigen receptor on B lymphocytes mediating early events in antigen presentation and signal transduction. Wild-type human mIgM constructs transfected into the murine B-cell lymphoma A20 are expressed as transmembrane proteins with antigen presentation and signaling functions comparable to the endogenous mIgG2A; the transfected wild-type mIgM is internalized rapidly after anti-Ig cross-linking. Transfected constructs lacking the normal three-amino acid cytoplasmic tail are expressed exclusively as phosphatidylinositol-linked proteins, lack both antigen presentation and signal transduction functions, and are internalized slowly following anti-Ig binding. The molecular mass of the cytoplasmic tail-deleted phosphatidylinositol-linked Ig molecule is consistent with cleavage of the transmembrane residues during processing. Cytoplasmic domains may therefore regulate the mode of expression of membrane proteins and thereby influence their functional capabilities.

Animals↗
PubMed1991

Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant.

The ligand (KL) for the c-kit receptor is a growth factor encoded at the mouse steel (Sl) locus. KL exists in both cell surface and soluble forms, though little is known of the regulation and functional significance of these forms. We show here that tissue-specific alternative splicing gives two types of KL mRNA. Both encode a transmembrane domain, but in transfected cells one produced the soluble form of KL at relatively high levels, whereas the other preferentially gave the cell surface form. Cell surface KL not only stimulated proliferation, but also mediated cell-cell adhesion. The SId allele, which impairs development of hematopoietic cells, melanocytes, and germ cells, has a deletion in the KL gene removing the transmembrane and intracellular domains. Expression of a corresponding cDNA gave a soluble protein that stimulated cellular proliferation but was not associated with the cell surface. These results provide evidence that cell surface KL has a critical role in the intact organism.

Amino Acid Sequence↗
PubMed1991

Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor.

The role of low affinity, heparin-like binding sites for basic fibroblast growth factor (bFGF) was investigated in CHO cells mutant in their metabolism of glycosaminoglycans. Heparan sulfate-deficient mutants transfected to express a cloned mouse FGF receptor cDNA are not able to bind bFGF. It is demonstrated that free heparin and heparan sulfate can reconstitute a low affinity receptor that is, in turn, required for the high affinity binding of bFGF. These studies suggest that the low affinity receptor is an accessory molecule required for binding of bFGF to the high affinity site. Such an obligatory interaction of low and high affinity FGF receptors suggests a physiological role for heparin-like, low affinity receptors and constitutes a novel mechanism for the regulation of growth factor-receptor interactions.

Animals↗
PubMed1991

Binary system for regulating transgene expression in mice: targeting int-2 gene expression with yeast GAL4/UAS control elements.

We have developed a binary transgenic system that activates an otherwise silent transgene in the progeny of a simple genetic cross. The system consists of two types of transgenic mouse strains, targets and transactivators. A target strain bears a transgene controlled by yeast regulatory sequences (UAS) that respond only to the yeast transcriptional activator GAL4. A transactivator strain expresses an active GAL4 gene that can be driven by any selected promoter. The current paradigm uses the murine growth factor int-2 cDNA as the target gene and the GAL4 gene driven by the mouse mammary tumor virus long terminal repeat as the transactivator. Both target and transactivator strains are phenotypically normal. By contrast, the bigenic offspring of these two strains express high levels of the target int-2 gene in each organ expressing the GAL4 transactivator. They also display a characteristic dominant int-2 phenotype that consists of epithelial hyperplasia in mammary and salivary glands, as well as prostatic and epididymal hypertrophy, which results in male sterility.

Animals↗
PubMed1991

Close linkage of a transgene insertion site to the steel (Sl) locus on mouse chromosome 10.

The characterization of the insertion sites of exogenous sequences in transgenic mice can identify loci that are potentially useful for the genetic analysis of the mammalian genome. We have found that the transgene insertion site in the transgenic line TG.EB is tightly linked with the Steel (Sl) locus on mouse chromosome 10. In a backcross between doubly heterozygous transgenic Sl (Tg.EB +/+ Sl) mice and wild-type mice, only one recombinant was found in 135 progeny (recombination percentage = 0.7 +/- 0.7). The recombination frequency of the transgene with marker loci known to flank Sl was consistent with a map position close to Sl. Genomic sequences that are adjacent to the transgene insertion site were cloned and found to be tightly linked with the Sl locus in interspecific crosses using nontransgenic mice. Recombination analysis utilizing the transgene insertion site locus was used to show that a recently identified hematopoietic growth factor is encoded at Sl. The cloned sequences from the transgene insertion site are polymorphic in inbred strains of mice and can be utilized to determine the genotype at Sl during early embryonic development. Further, they may be useful in characterizing the genomic region near Sl that is affected in Sl deletion mutants.

Animals↗
PubMed1991

SV40 T antigen transforms calcitonin cells of the thyroid but not CGRP-containing neurons in transgenic mice.

Neurons are postmitotic for the adult life of animals. Tumors rarely, if ever, arise from neurons in the adult, although they do from other cells from the same lineage, such as the neuroendocrine C cells of the thyroid. We have found that 2 kb of the calcitonin gene-related peptide (CGRP)/calcitonin gene suffices to target expression to CGRP-containing neurons, such as those in the dorsal root ganglia (DRG), and to the calcitonin-secreting C cells of the thyroid. Using this promoter we have examined the effect of two potentially transforming oncogenes in these two different populations. Overexpression of c-myc for periods of up to two years does not transform either cell type, whereas SV40 Tag causes early onset medullary thyroid carcinoma, but does not transform the dorsal root ganglia neurons. This suggests that as part of the terminal differentiation process of these neurons, the cessation of mitosis is accompanied by a relative refractoriness to oncogenes that may transform other cells of the same lineage.

Animals↗
PubMed1991

A human gene homologous to the formin gene residing at the murine limb deformity locus: chromosomal location and RFLPs.

The murine limb deformity (ld) locus resides on mouse chromosome 2 and gives rise to a recessively inherited, characteristic limb deformity/renal aplasia phenotype. In this locus in the mouse, a gene, termed the "formin" gene, has been identified which encodes an array of differentially processed transcripts in both adult and embryonic tissues. A set of these transcripts are disrupted in independent mutant mouse ld alleles. We wish to report the isolation of a human genomic clone which is homologous to the mouse formin gene by virtue of sequence comparison and expression of conserved exons. Among human fetal tissues analyzed, the kidney appears to be a major site of expression. This human gene, LD, maps to chromosome 15q11----qter in mouse human somatic cell hybrids and, specifically, to 15q13----q14 by chromosomal in situ hybridization. This localization establishes both LD and beta 2-microglobulin as syntenic genes on mouse chromosome 2 and human chromosome 15 and implies the interspecies conservation of the region between them. In addition, we identify in the human locus two frequently occurring DNA polymorphisms which can be used to test the linkage of LD to known human dysmorphoses.

Alleles↗
PubMed1991

Transgenic oncogene mice. Tumor phenotype predicts genotype.

The hypothesis that oncogenes influence tumor phenotype was tested by examining slides from 607 mammary tumors from 407 transgenic mice bearing the ras, myc, and/or neu oncogenes. Most tumors (91%) had patterns (phenotypes) that could not be classified by Dunn's standard nomenclature. The nonstandard tumors were described as eosinophilic small cell (SC), basophilic large cell (LC), or pale intermediate cell (IC). The SC tumor was associated with ras, the LC was associated with myc, and the IC was associated with neu, with specificities more than .90 and sensitivities ranging from .99 to .48. Thus, the tumor phenotype could be used to predict which oncogene was present in the animal. The presence of myc in combination with either ras or neu resulted in the predominance of LC tumors and accelerated tumorigenesis. The combination of ras and neu resulted in a decreased tumor incidence. Thus, knowledge of the oncogenes that were present could be used to predict the natural history of the disease.

Animals↗
PubMed1990

Mutations of immunoglobulin transmembrane and cytoplasmic domains: effects on intracellular signaling and antigen presentation.

The membrane-bound form of immunoglobulin serves as an antigen-specific receptor for B cells mediating signal transduction and antigen presentation. We have developed an assay that reconstitutes both these physiologic responses with respect to the antigen phosphorylcholine. By introducing specific mutations in the human Ig mu chain gene, we have shown that certain transmembrane residues and the short cytoplasmic domain are crucial for these two activities. Moreover, elimination of a single transmembrane hydroxyl group severely inhibits antigen presentation without affecting signal transduction, suggesting that these two functions are mediated by different protein interactions.

Amino Acid Sequence↗
PubMed1990

The kit ligand: a cell surface molecule altered in steel mutant fibroblasts.

The c-kit proto-oncogene, the gene at the mouse W developmental locus, is one of a substantial group of genes that appear to encode cell surface receptors but for which the ligands are unknown. We have characterized the kit ligand by a generally applicable approach: the receptor extracellular domain was genetically fused to placental alkaline phosphatase, producing a soluble receptor affinity reagent with an enzyme tag that could be easily and sensitively traced. This fusion protein, APtag-KIT, was used to demonstrate a specific binding interaction (KD = 3 x 10(-8) M) with a ligand on 3T3 fibroblast lines. In situ staining showed labeling over the whole surface of the 3T3 cells, but not extending to adjacent nonexpressing cells. These findings provide direct molecular evidence that the kit ligand can exist as a cell surface protein. Binding was not detected on 3T3 fibroblasts carrying the steel (Sl) mutation, confirming the biological significance of the binding activity and demonstrating that mutations at the Sl locus affect the expression or structure of the kit ligand.

Animals↗
PubMed1990

The hematopoietic growth factor KL is encoded by the Sl locus and is the ligand of the c-kit receptor, the gene product of the W locus.

Mutations at the steel locus (Sl) of the mouse affect the same cellular targets as mutations at the white spotting locus (W), which is allelic with the c-kit proto-oncogene. We show that KL, a hematopoietic growth factor obtained from conditioned medium of BALB/c 3T3 fibroblasts that stimulates the proliferation of mast cells and early erythroid progenitors, specifically binds to the c-kit receptor. The predicted amino acid sequence of isolated KL-specific cDNA clones suggests that KL is synthesized as an integral transmembrane protein. Linkage analysis maps the KL gene to the Sl locus on mouse chromosome 10, and KL sequences are deleted in the genome of the Sl mouse. These results indicate that the Sl locus encodes the ligand of the c-kit receptor, KL.

Amino Acid Sequence↗
PubMed1990

'Formins': proteins deduced from the alternative transcripts of the limb deformity gene.

Vertebrate limb formation is an evolutionarily conserved process programmed by an array of morphogenetic genes. As a result of transgene insertion, we previously identified a mutation at the mouse limb deformity (ld) locus that disrupts embryonic pattern formation, resulting in a reduction and fusion of the distal bones and digits of all limbs as well as variable incidence of renal aplasia. We have now characterized the ld locus at the molecular level. It contains evolutionarily conserved coding sequences that are transcribed in adult and embryonic tissues as a complex group of low abundance messenger RNAs created by alternative splicing and differential polyadenylation. The association of these transcripts with the gene responsible for the mutant phenotype was established by demonstrating that they are disrupted in two independently arising ld alleles. We have now deduced the structure of several novel proteins (termed formins) from the long open reading frames encoded by the various ld transcripts. The observation of these different RNA transcripts in different tissues suggests that the formins play a part in the formation of several organ systems.

Alleles↗
PubMed1990

Disruption of formin-encoding transcripts in two mutant limb deformity alleles.

The recent identification of a gene residing at the mouse limb deformity (ld) locus permits us to test the hypothesis that disruption of this gene is responsible for an inherited anomaly affecting embryonic pattern formation. The gene gives rise to alternatively processed messenger RNAs that can be translated as a family of related protein products, termed the formins. We have now analysed transcripts from this gene in four independently isolated mutant alleles. In two of these, the ldHd allele (created by insertion of a transgene) and the ldIn2 allele (created by a translocation-inversion involving mouse chromosomes 2 and 17), a common subset of ld transcripts is abolished, but others are apparently unaltered. The correlation of altered transcripts in two independent ld mutants strongly supports the notion that one or more altered formins is responsible for the observed phenotype. That the defect is limited to the limb and kidney, despite expression of ld mRNA in other unaffected organs, suggests that these mutant alleles represent only partial loss of ld function.

Alleles↗
PubMed1990

IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice.

We have assessed the biologic role of IL-4 by fusing its gene to an immunoglobulin promoter/enhancer and introducing it into transgenic mice. By attenuating the transgene promoter through the insertion of E. coli lac operator sequences, we have created a series of animals that constitutively express varying amounts of IL-4. Overexpression of IL-4 results in a marked increase in serum IgE levels and the appearance of an inflammatory ocular lesion (blepharitis) with characteristic histopathologic features seen in allergic reactions. In addition, expression of the IL-4 transgene in the thymus perturbs T cell maturation, reducing the population of immature CD4+CD8+ thymocytes and peripheral T cells while increasing the population of mature CD8+ thymocytes. These results demonstrate that deregulation of a single cytokine gene in vivo can induce a complex inflammatory reaction resembling that observed in human allergic disease.

Animals↗
PubMed1990

Structure and expression of the human immunoglobulin lambda genes.

We determined the DNA sequence of two large regions of chromosome 22: 33.7 kb containing the C lambda complex; and 5.2 kb 5' of the functionally rearranged lambda gene from the human myeloma, U266. Analysis of these sequences reveals the complete structure of the human C lambda complex and a previously undescribed seventh C lambda region that may encode the Ke+Oz- lambda protein. The seven constant regions are organized in a tandem array, and each is preceded by a single J lambda region. lambda 1, lambda 2, lambda 3, and lambda 7 are apparently active genes, while lambda 4, lambda 5, and lambda 6 are pseudogenes. There are no other J lambda or C lambda regions within a 60-kb region surrounding the C lambda complex; however, there are at least four other lambda-like genes and lambda pseudogenes in the human genome. The lambda genes appear to have evolved via a series of gene duplication events resulting from unequal crossing over or gene conversion between the highly conserved C lambda regions on mispaired chromosomes. The lack of Alu sequences in this large segment of DNA suggests that the C lambda complex resulted from a recent amplification of a smaller Alu-free segment of DNA. Illegitimate recombination between repeated sequences containing lambda 2 and lambda 3 may be responsible for variable amplification of the lambda genes. We also found a 1,377-bp open reading frame (ORF) located on the opposite strand in the region containing lambda 7. While this ORF is flanked by potential RNA splicing signals, we have no evidence that it is part of a functional gene. We also discovered a V lambda pseudogene, called psi V lambda 1, 3 kb upstream of the U266 lambda gene. Using primer extension analysis to map the transcription start in the human lambda gene, we have identified its initiation point 41 bp upstream of the initiation codon. Analysis of the lambda promoter reveals that it contains a TATAA box at position -29 relative to the transcription initiation site and an octamer sequence at -67. Computer analysis of 40 kb of DNA sequences surrounding the human lambda locus has revealed no sequences resembling the kappa or IgH transcriptional enhancers, nor have in vitro analyses for function revealed enhancer activity. A comparison of these results with those obtained in separate studies with transgenic mice point to a complex, developmentally linked mechanism of transcriptional activation.

Amino Acid Sequence↗