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1.
Nucleic Acids Res ; 31(3): 922-34, 2003 Feb 01.
Article in English | MEDLINE | ID: mdl-12560488

ABSTRACT

Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome DeltaYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from DeltaYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.


Subject(s)
Centromere/genetics , Chromosomes, Artificial, Human , Cloning, Molecular/methods , Recombination, Genetic , Saccharomyces cerevisiae/genetics , Base Sequence , Cell Line , Centromere/chemistry , Humans , Kinetochores/chemistry , Models, Genetic , Molecular Sequence Data , Sequence Analysis, DNA , Transformation, Genetic
2.
EMBO Rep ; 2(10): 910-4, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11571265

ABSTRACT

We have investigated the potential of PAC-based vectors as a route to the incorporation of a gene in a mammalian artificial chromosome (MAC). Previously we demonstrated that a PAC (PAC7c5) containing alpha-satellite DNA generated mitotically stable MACs in human cells. To determine whether a functional HPRT gene could be assembled in a MAC, PAC7c5 was co-transfected with a second PAC containing a 140 kb human HPRT gene into HPRT-deficient HT1080 cells. Lines were isolated containing a MAC hybridizing with both alpha-satellite and HPRT probes. The MACs segregated efficiently, associated with kinetochore proteins and stably expressed HPRT message after 60 days without selection. Complementation of the parental HPRT deficiency was confirmed phenotypically by growth on HAT selection. These results suggest that MACs could be further developed for delivering a range of genomic copies of genes into cells and that stable transgene expression can be achieved.


Subject(s)
Chromosomes, Artificial , Gene Expression , Genetic Techniques , Hypoxanthine Phosphoribosyltransferase/genetics , Blotting, Northern , Cell Division , Cell Line , DNA, Complementary/metabolism , Humans , In Situ Hybridization, Fluorescence , Microsatellite Repeats , Microscopy, Fluorescence , Mitosis , Phenotype , Time Factors , Transfection , Transgenes
3.
Peptides ; 22(3): 325-34, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11287086

ABSTRACT

Neuropeptide Y (NPY) from the brain of an amphibian from the order Gymnophiona (the caecilian, Typhlonectes natans) was characterized. We cloned a 790 base pair cDNA encoding the caecilian NPY precursor. The open reading frame consisted of 291 bases, indicating an NPY precursor of 97 amino acids. Both deduced and isolated NPY primary structures were Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu(10)-Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-Lys-Tyr(20)-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu(30)-Ile-Thr-Arg-Gln-Arg-Tyr. NH2. In caecilian brain, we observed NPY immunoreactive cells within the medial pallium, basal forebrain, preoptic area, midbrain tegmentum and trigeminal nucleus. The prevalence of preoptic and hypothalamic terminal field staining supports the hypothesis that NPY controls pituitary function in this caecilian.


Subject(s)
Brain/metabolism , Neuropeptide Y/biosynthesis , Neuropeptide Y/chemistry , Amino Acid Sequence , Amino Acids/chemistry , Amphibians , Animals , Base Sequence , Brain/pathology , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA, Complementary/metabolism , Humans , Molecular Sequence Data , Neuropeptide Y/genetics , Open Reading Frames , Time Factors , Tissue Distribution
4.
Hum Mol Genet ; 9(11): 1623-31, 2000 Jul 01.
Article in English | MEDLINE | ID: mdl-10861289

ABSTRACT

Mammalian artificial chromosomes (MACs) form in HT1080 cells after transfecting linear yeast artificial chromosome constructs minimally containing competent alphoid arrays, a selectable marker and terminal human telomere repeats. Restrictions on the structure of input DNA in MAC formation were investigated by transfecting circular or linear alphoid constructs with or without human telomere arrays and by varying the position and orientation of the telomere arrays on input linear constructs. Circular input DNA efficiently produced MACs. Absence of telomere arrays from circular input molecules did not significantly alter MAC formation rates. Linear constructs capped with telomere arrays generated MACs effectively, but a severe reduction in MAC formation was observed from linear constructs lacking telomere arrays. Human telomere arrays positioned 1-5 kb from linear construct ends and in either orientation were able to promote MAC formation with similar efficiencies. Both circular and linear input constructs generated artificial chromosomes that efficiently segregated in the absence of selection. Telomeres were not detected on the MACs, regardless of the inclusion of telomere arrays on input DNA, suggesting that circular chromosomes were formed. We found no evidence for acquisition of host cell DNA, which is consistent with de novo chromosome assembly.


Subject(s)
Chromosomes, Human/genetics , DNA, Satellite/genetics , Telomere/genetics , Bacteriophage P1/genetics , Chromosomes, Artificial, Yeast , DNA, Circular/genetics , Humans , In Situ Hybridization, Fluorescence , Repetitive Sequences, Nucleic Acid , Tumor Cells, Cultured
5.
Brain Behav Evol ; 55(1): 14-25, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10773622

ABSTRACT

The molecular forms and brain distribution of gonadotropin-releasing hormone (GnRH) have been well studied in the amphibian orders Urodela (salamanders and newts) and Anura (frogs and toads). In the order Gymnophiona (caecilians), however, few species have been investigated. Antibodies against different molecular forms of GnRH were used to immunohistochemically localize the GnRH-containing neurons in the brain of the caecilian, Typhlonectes natans which differs from most other amphibians in that it is viviparous. An antibody selective for mammalian GnRH recognized cell bodies predominantly in the septo-preoptic area but only with occasional cell bodies in the lateral hypothalamus and ventral thalamic eminence. Thick, prominent fibers in the septal region and fibers within the terminal nerve were also labeled. An antibody selective for chicken-II GnRH labeled a population of cell bodies in the dorsal hypothalamus, ventral thalamus and midbrain tegmentum. Thin fibers projected laterally from these cells. An antibody specific for salmon GnRH did not label cell bodies but did show intense terminal field immunoreactivity. The brain of this caecilian, therefore, contains three antigenically distinct forms of GnRH. The mammalian and chicken-II GnRH peptides have been shown in other amphibians but the distribution of cells and fibers was unique in this caecilian.


Subject(s)
Amphibians/physiology , Brain Chemistry/physiology , Brain/anatomy & histology , Gonadotropin-Releasing Hormone/metabolism , Animals , Antibody Specificity , Chickens/immunology , Female , Immunohistochemistry , Male , Mammals/immunology , Nerve Fibers/metabolism , Nerve Fibers/physiology , Salmon/immunology , Species Specificity
6.
Mamm Genome ; 10(7): 662-9, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10384037

ABSTRACT

qkI, encoding a KH domain-containing RNA binding protein, has been isolated as a candidate gene for the mouse neurological mutation quaking. Here, we describe detailed studies on its genomic structure and expression pattern. We isolated approximately 1 Mb of genomic region containing the quaking locus and determined its genomic organization. The qkI locus contains at least 9 exons spanning approximately 65 kb of DNA. It gives rise to six distinct transcripts encoding, theoretically, five different protein isoforms. Exons 1 through 4 are shared by all the transcripts, whereas coding exons and two distinct 3'-UTRs downstream to the exon 4 are differentially utilized. One isoform has a truncated KH domain and may act as an antagonist to the others. These findings and identification of a single transcription initiation site suggest that differential expression of each transcript is regulated by alternative splicing. Expression of each alternative transcript and protein product was also examined. Two types of transcripts, 5 kb-A and B, are most abundant in the brain of newborn mice and are gradually downregulated thereafter. In contrast, the other three messages, 6 kb, 7 kb-A and B, increase as myelination proceeds and peak at 2 weeks of age, corresponding to the most active stage of myelination. Although the qkI messages and their products are abundant in brain and heart, a lower level of expression was found in various other tissues tested. Alternative transcripts that share the same 3'-UTR showed very similar expression patterns, suggesting a regulatory role of the 3'-UTRs in qkI gene expression.


Subject(s)
RNA-Binding Proteins/genetics , 3' Untranslated Regions , Amino Acid Sequence , Animals , Base Sequence , Chromosomes, Artificial, Yeast , Cloning, Molecular , DNA Primers , Exons , Introns , Mice , Mice, Quaking , Molecular Sequence Data , Protein Biosynthesis , Transcription, Genetic
7.
Gene ; 188(2): 199-206, 1997 Apr 01.
Article in English | MEDLINE | ID: mdl-9133592

ABSTRACT

Mutations in the mouse quaking locus can result in two different types of developmental phenotypes: (1) a deficiency of myelin in the central nervous system that is accompanied by a characteristic tremor, or (2) embryonic lethality around day 9 of gestation. A quaking candidate gene (qkI) that encodes a KH motif protein has recently been identified. We have isolated and characterized cDNAs encoding the Xenopus quaking homologue (Xqua) and also assembled an almost complete human quaking sequence from expressed sequence tags. Sequence comparisons show that the amphibian and mammalian quaking transcripts exhibit striking conservation, both within the coding region and, unexpectedly, in the 3' UTR. Two Xqua transcripts 5 kb and 5.5 kb in length are differentially expressed in the Xenopus embryo, with the 5 kb transcript being detected as early as the gastrula stage of development. Using an in vitro assay, we have demonstrated RNA-binding activity for quaking protein encoded by the 5 kb transcript. Overall, the high sequence conservation of quaking sequences suggests an important conserved function in vertebrate development, probably in the regulation of RNA metabolism.


Subject(s)
RNA-Binding Proteins/genetics , Xenopus Proteins , Amino Acid Sequence , Animals , Base Sequence , Conserved Sequence , DNA, Complementary , Embryo, Nonmammalian/metabolism , Gene Expression , Humans , Mice , Molecular Sequence Data , RNA/metabolism , RNA-Binding Proteins/metabolism , Xenopus
8.
J Neurosci ; 16(24): 7941-9, 1996 Dec 15.
Article in English | MEDLINE | ID: mdl-8987822

ABSTRACT

qkI, a newly cloned gene lying immediately proximal to the deletion in the quakingviable mutation, is transcribed into three messages of 5, 6, and 7 kb. Antibodies raised to the unique carboxy peptides of the resulting QKI proteins reveal that, in the nervous system, all three QKI proteins are expressed strongly in myelin-forming cells and also in astrocytes. Interestingly, individual isoforms show distinct intracellular distributions: QKI-6 and QKI-7 are localized to perikaryal cytoplasm, whereas QKI-5 invariably is restricted to the nucleus, consistent with the predicted role of QKI as an RNA-binding protein. In quakingviable mutants, which display severe dysmyelination, QKI-6 and QKI-7 are absent exclusively from myelin-forming cells. By contrast, QKI-5 is absent only in oligodendrocytes of severely affected tracts. These observations implicate QKI proteins as regulators of myelination and reveal key insights into the mechanisms of dysmyelination in the quakingviable mutant.


Subject(s)
Neurons/metabolism , RNA-Binding Proteins/metabolism , Aging/metabolism , Animals , Astrocytes/metabolism , Brain/cytology , Brain/metabolism , Demyelinating Diseases/genetics , Demyelinating Diseases/metabolism , Immune Sera , Isomerism , Mice , Mice, Quaking , Myelin Sheath/physiology , Neuroglia/metabolism , Oligodendroglia/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/immunology , Schwann Cells/metabolism , Schwann Cells/physiology
9.
Nat Genet ; 12(3): 260-5, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8589716

ABSTRACT

The mouse quaking gene, essential for nervous system myelination and survival of the early embryo has been positionally cloned. Its sequence implies that the locus encodes a multifunctional gene used in a specific set of developing tissues to unite signal transduction with some aspect of RNA metabolism. The quaking(viable) (qkv) mutation has one class of messages truncated by a deletion. An independent ENU-induced mutation has a nonconservative amino acid change in one of two newly identified domains that are conserved from the C. elegans gld-1 tumour suppressor gene to the human Src-associated protein Sam68. The size and conservation of the quaking gene family implies that the pathway defined by this mutation may have broad relevance for rapid conveyance of extracellular information directly to primary gene transcripts.


Subject(s)
Embryonic and Fetal Development/genetics , Myelin Sheath/physiology , RNA-Binding Proteins/genetics , Signal Transduction , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cloning, Molecular/methods , DNA Primers , DNA-Binding Proteins/chemistry , Embryonic and Fetal Development/physiology , Humans , Mice , Mice, Inbred DBA , Mice, Quaking , Molecular Sequence Data , Mutation , Nervous System/embryology , Phosphoproteins/chemistry , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/physiology , Restriction Mapping
10.
Genetics ; 131(1): 175-82, 1992 May.
Article in English | MEDLINE | ID: mdl-1350556

ABSTRACT

Many mutations affecting mouse development have been mapped to the t-complex of mouse chromosome 17. We have obtained 17 cosmid clones as molecular markers for this region by screening a hamster-mouse chromosome 17 and 18 cell hybrid cosmid library with mouse-specific repetitive elements and mapping positive clones via t-haplotype vs. C3H restriction fragment length polymorphism (RFLP) analysis. Twelve of the clones mapping distal to Leh66B in t-haplotypes are described here. Using standard RFLP analysis or simple sequence length polymorphism between t-haplotypes, exceptional partial t-haplotypes and nested sets of inter-t-haplotype recombinants, five cosmids have been mapped in or around In(17)3 and seven in the most distal inversion In17(4). More precise mapping of four of the cosmids from In(17)4 shows that they will be useful in the molecular identification of some of the recessive lethals mapped to the t-complex: two cosmids map between H-2K and Crya-1, setting a distal limit in t-haplotypes for the position of the tw5 lethal, one is inseparable from the tw12 lethal, and one maps distal to tf near the t0(t6) lethal and cld.


Subject(s)
Genetic Markers , Mutation , Animals , Base Sequence , Chromosome Mapping , Cosmids , Electrophoresis, Agar Gel , Genes, Lethal , Genes, Recessive , Haplotypes , Mice , Mice, Inbred C3H , Molecular Sequence Data , Nucleic Acid Hybridization , Polymorphism, Restriction Fragment Length , Recombination, Genetic
11.
Genetics ; 131(1): 183-90, 1992 May.
Article in English | MEDLINE | ID: mdl-1592235

ABSTRACT

Five randomly identified cosmids have been mapped proximal to the Leh66D locus on mouse chromosome 17. Two of these cosmids, Au10 and Au119, map near the neurological mutation quaking. Au119 is deleted in qkviable/qkviable DNA, whereas Au10 is not. Au76 maps to a gene-rich region near the Time locus. The Au76 locus encodes a member of a low copy gene family expressed in embryos, the adult central nervous system and testis. A second member of this family has been mapped to chromosome 15 near c-sis (PDGF-B). At the centromeric end of chromosome 17, Au116 maps near the Tu1 locus, and along with Au217rs identifies a region of unusually high recombinational activity between t-haplotypes and wild-type chromosomes. Au217I and II map to the large inverted repeats found at the proximal end of the wild-type chromosome. In addition, the Au217I and/or II loci encode testis transcripts not expressed from t-haplotypes.


Subject(s)
Chromosome Deletion , Chromosomes , Genetic Markers , Mice, Quaking/genetics , Animals , Central Nervous System , Chromatin , Chromosome Mapping , Cosmids , Embryo, Mammalian , Haplotypes , Male , Mice , Multigene Family , Mutation , Recombination, Genetic , Testis , Transcription, Genetic
12.
Genetics ; 126(4): 1093-102, 1990 Dec.
Article in English | MEDLINE | ID: mdl-2076812

ABSTRACT

We describe here the identification and genomic organization of mouse t haplotype-specific elements (TSEs) 7.8 and 5.8 kb in length. The TSEs exist as submegabase-long clusters of tandem repeats localized in the Tla region of the major histocompatibility complex of all t haplotype chromosomes examined. In contrast, no such clusters were detected among 12 inbred strains of Mus musculus and other Mus species; thus, clusters of TSEs represent the first absolutely qualitative difference between t haplotypes and wild-type chromosomes. Pulsed field gel electrophoresis shows that the number of clusters, and the number of repeats in each cluster are extremely variable. Dramatic quantitative differences of TSEs uniquely distinguish every independent t haplotype from any other. The complete nucleotide sequence of one 7.8-kb TSE reveals significant homology to the ETn (a major transcript in the early embryo of the mouse), and some homologies to intracisternal A-particles and the mammary tumor virus env gene. Apart from the diagnostic relevance to t haplotypes, evolutionary and functional significances are discussed with respect to chromosome structure and genetic recombination.


Subject(s)
Major Histocompatibility Complex/genetics , Multigene Family , Repetitive Sequences, Nucleic Acid , Animals , Base Sequence , Blotting, Southern , Haplotypes , Mice , Mice, Inbred C3H , Molecular Sequence Data , Restriction Mapping , Sequence Homology, Nucleic Acid
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