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W J Pavan

Publications and source records attributed to W J Pavan.

34 records · Page 2Linked to original sources
PubMed1997

Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene.

An integrated human-mouse positional candidate approach was used to identify the gene responsible for the phenotypes observed in a mouse model of Niemann-Pick type C (NP-C) disease. The predicted murine NPC1 protein has sequence homology to the putative transmembrane domains of the Hedgehog signaling molecule Patched, to the cholesterol-sensing regions of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and SREBP cleavage-activating protein (SCAP), and to the NPC1 orthologs identified in human, the nematode Caenorhabditis elegans, and the yeast Saccharomyces cerevisiae. The mouse model may provide an important resource for studying the role of NPC1 in cholesterol homeostasis and neurodegeneration and for assessing the efficacy of new drugs for NP-C disease.

Amino Acid Sequence↗
PubMed1997

Substantial narrowing of the Niemann-Pick C candidate interval by yeast artificial chromosome complementation.

Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.

Animals↗
PubMed1997

Genomic organization of the murine Miller-Dieker/lissencephaly region: conservation of linkage with the human region.

Several human syndromes are associated with haploinsufficiency of chromosomal regions secondary to microdeletions. Isolated lissencephaly sequence (ILS), a human developmental disease characterized by a smooth cerebral surface (classical lissencephaly) and microscopic evidence of incomplete neuronal migration, is often associated with small deletions or translocations at chromosome 17p13.3. Miller-Dieker syndrome (MDS) is associated with larger deletions of 17p13.3 and consists of classical lissencephaly with additional phenotypes including facial abnormalities. We have isolated the murine homologs of three genes located inside and outside the MDS region: Lis1, Mnt/Rox, and 14-3-3 epsilon. These genes are all located on mouse chromosome 11B2, as determined by metaphase FISH, and the relative order and approximate gene distance was determined by interphase FISH analysis. The transcriptional orientation and intergenic distance of Lis1 and Mnt/Rox were ascertained by fragmentation analysis of a mouse yeast artificial chromosome containing both genes. To determine the distance and orientation of 14-3-3 epsilon with respect to Lis1 and Mnt/Rox, we introduced a super-rare cutter site (VDE) that is unique in the mouse genome into 14-3-3 epsilon by gene targeting. Using the introduced VDE site, the orientation of this gene was determined by pulsed field gel electrophoresis and Southern blot analysis. Our results demonstrate that the MDS region is conserved between human and mouse. This conservation of linkage suggests that the mouse can be used to model microdeletions that occur in ILS and MDS.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1997

Melanocyte development in vivo and in neural crest cell cultures: crucial dependence on the Mitf basic-helix-loop-helix-zipper transcription factor.

The more than 20 different Mitf mutations in the mouse are all associated with deficiencies in neural crest-derived melanocytes that range from minor functional disturbances with some alleles to complete absence of mature melanocytes with others. In the trunk region of wild-type embryos, Mitf-expressing cells that coexpressed the melanoblast marker Dct and the tyrosine kinase receptor Kit were found in the dorsolateral neural crest migration pathway. In contrast, in embryos homozygous for an Mitf allele encoding a non-functional Mitf protein, Mitf-expressing cells were extremely rare, no Dct expression was ever found, and the number of Kit-expressing cells was much reduced. Wild-type neural crest cell cultures rapidly gave rise to cells that expressed Mitf and coexpressed Kit and Dct. With time in culture, Kit expression was increased, and pigmented, dendritic cells developed. Addition of the Kit ligand Mgf or endothelin 3 or a combination of these factors all rapidly increased the number of Dct-positive cells. Cultures from Mitf mutant embryos initially displayed Mitf-positive cells similar in numbers and Kit-expression as did wild-type cultures. However, Kit expression did not increase with time in culture and the mutant cells never responded to Mgf or endothelin 3, did not express Dct, and never showed pigment. In fact, even Mitf expression was rapidly lost. The results suggest that Mitf first plays a role in promoting the transition of precursor cells to melanoblasts and subsequently, by influencing Kit expression, melanoblast survival.

Amino Acid Sequence↗
PubMed1995

A high-resolution linkage map of the lethal spotting locus: a mouse model for Hirschsprung disease.

Mice homozygous for the lethal spotting (ls) mutation exhibit aganglionic megacolon and a white spotted coat owing to a lack of neural crest-derived enteric ganglia and melanocytes. The ls mutation disrupts the migration, differentiation, or survival of these neural crest lineages during mammalian development. A human congenital disorder, Hirschsprung disease (HSCR), is also characterized by aganglionic megacolon of the distal bowel and can be accompanied by hypopigmentation of the skin. HSCR has been attributed to multiple loci acting independently or in combination. The ls mouse serves as one animal model for HSCR, and the ls gene may represent one of the loci responsible for some cases of HSCR in humans. This study uses 753 N2 progeny from a combination of three intersubspecific backcrosses to define the molecular genetic linkage map of the ls region and to provide resources necessary for positional cloning. Similar to some cases of HSCR, the ls mutation acts semidominantly, its phenotypic effects dependent upon the presence of modifier genes segregating in the crosses. We have now localized the ls mutation to a 0.8-cM region between the D2Mit113 and D2Mit73/D2Mit174 loci. Three genes, endothelin-3 (Edn3), guanine nucleotide-binding protein alpha-stimulating polypeptide 1 (Gnas), and phosphoenolpyruvate carboxykinase (Pck1) were assessed as candidates for the ls mutation. Only Edn3 and Gnas did not recombine with the ls mutation. Mutational analysis of the Edn3 and Gnas genes will determine whether either gene is responsible for the neural crest deficiencies observed in ls/ls mice.

Animals↗
PubMed1995

Quantitative trait loci that modify the severity of spotting in piebald mice.

Mice homozygous for the recessive mutation piebald (s) exhibit a white-spotted coat caused by the defective development of neural crest-derived melanocytes. The severity of white spotting varies greatly, depending on the genetic background on which s is expressed. A backcross between two inbred strains of s/s mice that exhibit large differences in the degree of spotting was used to identify six genetic modifiers of piebald spotting on chromosomes 2, 5, 7, 8, 10, and 13. The loci differed in their spatial contribution to spotting on the dorsal versus ventral surfaces of mice; nonadditive interactions were observed between loci on chromosomes 2 and 5. This study underscores the power of using genetic analyses to identify and analyze loci involved in modifying the severity of phenotypic traits in mice.

Animals↗
PubMed1994

Piebald lethal (sl) acts early to disrupt the development of neural crest-derived melanocytes.

Mice homozygous for the piebald lethal (sl) mutation have a predominantly white coat due to the absence of neural crest-derived melanocytes in the hair follicles. To investigate the time in embryonic development when the s1 gene affects the melanocyte lineage, we compared the distribution of melanocyte precursors in wild-type and mutant embryos, using an antibody specific for tyrosinase-related protein 2 (TRP-2). TRP-2 positive cells were first observed adjacent to the anterior cardinal vein in 10.5-day postcoitem wild-type embryos. From 11.5 to 13.5 days postcoitem, there was a nonuniform distribution of TRP-2 positive cells along the anterior-posterior axis, with the highest density of cells in the head and tail regions. Along the dorsal-ventral axis, the cells were restricted to positions lateral, but never dorsal, to the neural tube. In homozygous sl/sl embryos TRP-2 staining was restricted to the non-neural crest-derived melanocytes of the pigmented retinal epithelium and the telencephalon. Few positive cells were seen in areas that will form neural crest-derived melanocytes in the inner ear, skin, hair follicles, leg musculature, or heart. We conclude that the piebald lethal mutation acts prior to the onset of TRP-2 expression to disrupt the development of neural crest-derived melanocytes. The non-uniform distribution of melanoblasts in wild-type mice suggests that piebald acts stochastically to affect melanocyte development.

Animals↗
PubMed1991

High-efficiency yeast artificial chromosome fragmentation vectors.

Chromosome fragmentation vectors (CFVs) are used to create deletion derivatives of large fragments of human DNA cloned as yeast artificial chromosomes (YACs). CFVs target insertion of a telomere sequence into the YAC via homologous recombination with Alu repetitive elements. This event results in the loss of all YAC sequences distal to the site of integration. A new series of CFVs has been developed. These vectors target fragmentation to both Alu and LINE human repetitive DNA elements. Recovery of deletion derivatives is ten- to 20-fold more efficient with the new vectors than with those described previously.

Chromosomes, Fungal↗
PubMed1991

Integrative selection of human chromosome-specific yeast artificial chromosomes.

Human specific "integrative selection vectors" (ISVs) were designed to optimize integration of a yeast-selectable marker specifically into yeast artificial chromosomes (YACs) derived from human but not mouse DNA. ISVs were transformed into a YAC genomic library constructed from DNA of a human-mouse somatic cell hybrid containing chromosome 21 (HSA21) as the only human chromosome. One percent of the yeast in the original library contained HSA21-derived YACs; between 45% and 54% of the yeast recovered after transformation with ISV vectors contained human YACs. Integrative selection provides a rapid means of obtaining a highly enriched population of human chromosome-specific YACs by eliminating the labor-intensive steps of isolating and screening primary transformants. The procedure is biased toward the selection of YACs that contain a large number of targets for homologous recombinations; thus, libraries constructed by this procedure will be composed primarily of the largest YACs in the population.

Chromosomes, Fungal↗
PubMed1990

Generation of deletion derivatives by targeted transformation of human-derived yeast artificial chromosomes.

Mammalian DNA segments cloned as yeast artificial chromosomes (YACs) can be manipulated by DNA-mediated transformation when placed in an appropriate yeast genetic background. A "fragmenting vector" has been developed that can introduce a yeast telomere and selectable marker into human-derived YACs at specific sites by means of homologous recombination, deleting all sequences distal to the recombination site. A powerful application of the method uses a human Alu family repeat sequence to target recombination to multiple independent sites on a human-derived YAC. Sets of deletion derivatives generated by this procedure greatly facilitate restriction mapping of large genomic segments. Targeting recombination with single copy sequences, such as cDNAs, will have many additional applications. This approach establishes a paradigm for manipulation and characterization of mammalian DNA segments cloned as YACs.

Chromosome Deletion↗
PubMed1990

Modification and transfer into an embryonal carcinoma cell line of a 360-kilobase human-derived yeast artificial chromosome.

A neomycin resistance cassette was integrated into the human-derived insert of a 360-kilobase yeast artificial chromosome (YAC) by targeting homologous recombination to Alu repeat sequences. The modified YAC was transferred into an embryonal carcinoma cell line by using polyethylene glycol-mediated spheroplast fusion. A single copy of the human sequence was introduced intact and stably maintained in the absence of selection for over 40 generations. A substantial portion of the yeast genome was retained in hybrids in addition to the YAC. Hybrid cells containing the YAC retained the ability to differentiate when treated with retinoic acid. This approach provides a powerful tool for in vitro analysis because it can be used to modify any human DNA cloned as a YAC and to transfer large fragments of DNA intact into cultured mammalian cells, thereby facilitating functional studies of genes in the context of extensive flanking DNA sequences.

Animals↗
PubMed1989

The mouse neurological mutant weaver maps within the region of chromosome 16 that is homologous to human chromosome 21.

Utilizing the backcross C57BL/6 wv/wv x (C57BL/6 wv/wv x MOLD/Rk), the mouse neurological mutation weaver (wv) was mapped less than 1 cM proximal to Ets-2 and Mx on mouse chromosome 16 (0.96 +/- 0.1% recombination). This region is known to include eight genes that are found on human chromosome 21 (HSA 21) and appears to be highly conserved between the two species. We therefore predict that the normal human homolog of wv will be located on HSA 21 and would be in dosage imbalance in individuals with Down syndrome.

Alleles↗
PubMed1988

Genetic mapping of the Mx influenza virus resistance gene within the region of mouse chromosome 16 that is homologous to human chromosome 21.

A total of 318 progeny from four backcrosses involving different laboratory strains and subspecies of Mus musculus were analyzed to map the Mx gene to the region of mouse chromosome 16 (MMU 16) which is homologous to human chromosome 21 (HSA 21). This result suggests that Mx will be found in the region of HSA 21 which has been implicated in Down syndrome when inherited in three copies.

Animals↗