Biomedical subjects

R K Moyzis

Publications and source records attributed to R K Moyzis.

At least 19 recordsLinked to original sources

PubMed2005

Sequence variants of the DRD4 gene in autism: further evidence that rare DRD4 7R haplotypes are ADHD specific.

A high prevalence of rare dopamine receptor D4 (DRD4) alleles in children diagnosed with attention-deficit hyperactivity disorder (ADHD) has been reported [Grady et al., 2003]. In this prior study, extensive resequencing/haplotype data of the DRD4 locus was used to suggest that population stratification was not the explanation for the high prevalence of rare alleles. In the current study, DNA resequencing/haplotyping was conducted on 136 DRD4 alleles obtained from autism probands, collected from the same geographic population as the prior ADHD probands (Orange County, CA). A number of studies have suggested that the susceptibility genes underlying these two disorders might partially overlap. Rare DRD4 variants were not uncovered in this autism sample beyond that expected by chance. These results suggest strongly that the high prevalence of rare DRD4 alleles in ADHD probands is due to ascertainment of the sample by diagnosis of ADHD.

Alleles↗

PubMed2004

The genetic architecture of selection at the human dopamine receptor D4 (DRD4) gene locus.

Associations of the seven-repeat (7R) allele of the human dopamine receptor D4 (DRD4) gene with both the personality trait of novelty seeking and attention deficit/hyperactivity disorder have been reported. Recently, on the basis of the unusual DNA sequence organization of the DRD4 7R 48-bp tandem repeat (VNTR), we proposed that the 7R allele originated as a rare mutational event that increased to high frequency by positive selection. We now have resequenced the entire DRD4 locus from 103 individuals homozygous for 2R, 4R, or 7R variants of the VNTR, a method developed to directly estimate haplotype diversity. DNA from individuals of African, European, Asian, North and South American, and Pacific Island ancestry were used. 4R/4R homozygotes exhibit little linkage disequilibrium (LD) over the region examined, with more polymorphisms observed in DNA samples from African individuals. In contrast, the evidence for strong LD surrounding the 7R allele is dramatic, with all 7R/7R individuals (including those from Africa) exhibiting the same alleles at most polymorphic sites. By intra-allelic comparison at 18 high-heterozygosity sites spanning the locus, we estimate that the 7R allele arose prior to the upper Paleolithic era (approximately 40000-50000 years ago). Further, the pattern of recombination at these polymorphic sites is the pattern expected for selection acting at the 7R VNTR itself, rather than at an adjacent site. We propose a model for selection at the DRD4 locus consistent with these observed LD patterns and with the known biochemical and physiological differences between receptor variants.

Alleles↗

PubMed2003

High prevalence of rare dopamine receptor D4 alleles in children diagnosed with attention-deficit hyperactivity disorder.

Associations have been reported of the 7-repeat (7R) allele of the human dopamine receptor D4 (DRD4) gene with both the personality trait of novelty seeking and attention-deficit/hyperactivity disorder (ADHD). The increased prevalence of the 7R allele in ADHD probands is consistent with the common variant-common disorder hypothesis, which proposes that the high frequency of many complex genetic disorders is related to common DNA variants. Recently, based on the unusual DNA sequence organization and strong linkage disequilibrium surrounding the DRD4 7R allele, we proposed that this allele originated as a rare mutational event, which nevertheless increased to high prevalence in human populations by positive selection. We have now determined, by DNA resequencing of 250 DRD4 alleles obtained from 132 ADHD probands, that most ADHD 7R alleles are of the conserved haplotype found in our previous 600 allele worldwide DNA sample. Interestingly, however, half of the 24 haplotypes uncovered in ADHD probands were novel (not one of the 56 haplotypes found in our prior population studies). Over 10 percent of the ADHD probands had these novel haplotypes, most of which were 7R allele derived. The probability that this high incidence of novel alleles occurred by chance in our ADHD sample is much less than 0.0001. These results suggest that allelic heterogeneity at the DRD4 locus may also contribute to the observed association with ADHD.

Amino Acid Sequence↗

PubMed2001

A sequence-ready map of the human chromosome 1q telomere.

A 260-kb half-YAC clone derived from human chromosome 1q was mapped at high resolution using cosmid subclone fingerprint analysis and was integrated with overlapping clones from the telomeric end of a separately derived 1q44 BAC contig to create a sequence-ready map extending to the molecular telomere of 1q. Analysis of 100 kb of sample sequences from across the 260-kb region encompassed by the half-YAC revealed the presence of EST sequence matches corresponding to 12 separate Unigene clusters and to 12 separate unclustered EST sequences. Low-copy subtelomeric repeats typical of many human telomere regions are present within the distal-most 30 kb of 1q. The previously isolated and radiation hybrid-mapped markers Bda84F03, 1QTEL019, and WI11861 localized at distances approximately 32, 88, and 99 kb, respectively, from the 1q terminus. This sequence-ready map permits high-resolution integration of genetic maps with the DNA sequences directly adjacent to the tip of human chromosome 1q and will enable telomeric closure of the human chromosome 1q DNA reference sequence by connecting the molecular 1q telomere to an internal BAC contig.

Chromosomes, Artificial, Bacterial↗

PubMed2001

Integration of telomere sequences with the draft human genome sequence.

Telomeres are the ends of linear eukaryotic chromosomes. To ensure that no large stretches of uncharacterized DNA remain between the ends of the human working draft sequence and the ends of each chromosome, we would need to connect the sequences of the telomeres to the working draft sequence. But telomeres have an unusual DNA sequence composition and organization that makes them particularly difficult to isolate and analyse. Here we use specialized linear yeast artificial chromosome clones, each carrying a large telomere-terminal fragment of human DNA, to integrate most human telomeres with the working draft sequence. Subtelomeric sequence structure appears to vary widely, mainly as a result of large differences in subtelomeric repeat sequence abundance and organization at individual telomeres. Many subtelomeric regions appear to be gene-rich, matching both known and unknown expressed genes. This indicates that human subtelomeric regions are not simply buffers of nonfunctional 'junk DNA' next to the molecular telomere, but are instead functional parts of the expressed genome.

Chromosomes, Artificial, Bacterial↗

PubMed1999

Reduced telomere DNA content is correlated with genomic instability and metastasis in invasive human breast carcinoma.

Telomere shortening leads to genomic instability and has been correlated with poor outcome in several types of cancer. A recently described, robust titration assay was used to quantify telomere DNA content in frozen and paraffin-embedded specimens of 49 invasive human breast carcinomas, including tumors with normal or abnormal contents of genomic DNA, which produced regional, distant, or local disease. Telomere DNA contents ranged from 53% to 370% of the content in a reference DNA purified from normal placenta. Tumors were divided into three groups of approximately equal size based on increasing telomere DNA content. All of 16 tumors in the group with the least telomere DNA (Group I), were aneuploid compared to 9/17 tumors in the group with the most telomere DNA (Group III). The Chi-square test for trend indicated that tumors with the least telomere DNA were significantly more likely to be aneuploid than tumors with the most telomere DNA (p < 0.002). Twelve of 14 tumors in Group I also produced metastatic disease compared to 8/15 tumors in Group III. The Fischer Exact Test indicated that tumors with the least telomere DNA were significantly more likely to be metastatic than tumors with the most telomere DNA (p < 0.05). There was no association between telomere DNA content and patients' age, tumors' size, grade, stage, or fraction of cells in S-phase. The correlation of reduced telomere DNA content with aneuploidy and metastasis, both of which are associated with poor outcome in invasive breast carcinoma, implies that telomere DNA content also could have prognostic value.

Aneuploidy↗

PubMed1999

An integrated physical map for the short arm of human chromosome 5.

The short arm of human chromosome 5 contains approximately 48 Mb of DNA and comprises 1.5% of the genome. We have constructed a mega-YAC/ STS map of this region that includes 436 YACs anchored by 216 STSs. By combining and integrating our map with the 5p maps of other groups using the same recombinant DNA library, a comprehensive map was constructed that includes 552 YACs and 504 markers. The YAC map covers >94% of 5p in four YAC contigs, bridges the centromere, and includes an additional 5 Mb of 5q DNA. The average marker density is 95 kb. This integrated 5p map will serve as a resource for the continuing localization of genes on the short arm of human chromosome 5 and as a framework for both generating and aligning the DNA sequence of this region.

Animals↗

PubMed1998

Construction of human chromosome 16- and 5-specific circular YAC/BAC libraries by in vivo recombination in yeast (TAR cloning).

Transformation-associated recombination (TAR) in yeast was exploited for the selective isolation of human DNAs as large circular yeast artificial chromosomes (YACs) from two rodent/human hybrid cell lines containing human chromosomes 5 and 16. TAR cloning vectors containing the F-factor origin of replication were constructed for use in these experiments. Presence of the F-factor origin in TAR vectors provides the capability of transferring the YACs generated by in vivo recombination in yeast into Escherichia coli cells and propagating them as bacterial artificial chromosomes (BACs). A high enrichment of human versus rodent YACs was observed during isolation of human DNA from the rodent/human hybrid cell lines. Although <3% of the DNA content in the hybrid cells was human, as many as 75% of the transformants contained human YACs. In contrast to the standard YAC cloning method based on in vitro ligation, no human/mouse chimeras were observed during TAR cloning. The constructed human chromosome 16 YAC library had approximately 2.6x coverage, represented by 4320 YAC clones with an average insert size of 80 kb. YAC clones generated from chromosome 16 were successfully converted into BACs by electroporation of DNA isolated from yeast transformants into E. coli. The BAC clones represent approximately 0.6x chromosomal coverage. Pilot YAC and BAC libraries of chromosome 5 have been also constructed. The chromosomal distribution of YAC/BACs from chromosome 5 and chromosome 16 was evaluated by fluorescence in situ hybridization (FISH). The distribution of FISH signals appeared random along the length of each chromosome. We conclude that TAR cloning provides an efficient means for generating representative chromosome-specific YAC/BAC libraries.

Animals↗

PubMed1998

Centromeric protein B null mice are viable with no apparent abnormalities.

The centromere protein B (CENP-B) is a centromeric DNA/binding protein. It recognizes a 17-bp sequence motif called the CENP-B box, which is found in the centromeric region of most chromosomes. It binds DNA through its amino terminus and dimerizes through its carboxy terminus. CENP-B protein has been proposed to perform a vital role in organizing chromatin structures at centromeres. However, other evidence does not agree with this view. For example, CENP-B is found at inactive centromeres on stable dicentric chromosomes, and also mitotically stable chromosomes lacking alpha-satellite DNA have been reported. To address the biological function of CENP-B, we generated mouse null mutants of CENP-B by homologous recombination. Mice lacking CENP-B were viable and fertile, indicating that mice without CENP-B undergo normal somatic and germline development. Thus, both mitosis and meiosis are able to proceed normally in the absence of CENP-B.

Animals↗

PubMed1998

Inhibition of human telomerase by a retrovirus expressing telomeric antisense RNA.

Human telomerase, the RNA-dependent DNA polymerase that adds TTAGGG repeats to chromosome ends, is selectively expressed in immortalised cells and most tumours, suggesting a potential role for telomerase inhibitors in cancer therapy. Replication-deficient retroviruses were used to determine whether mRNA containing UUAGGG, the complementary sequence to the template region of the hTR telomerase RNA, is sufficient to inhibit telomerase activity. Telomerase activities measured by the telomeric repeat amplification protocol (TRAP) assay in extracts prepared from immortalised mouse fibroblasts, human HeLa cells and human kidney carcinoma cells were inhibited by 75% or greater in 26 of 56 cell clones expressing UUAGGG. Telomerase activity was not inhibited by expression of mRNA containing a transposed sequence, GGGAUU. Telomerase activities in vivo were inferred from changes in cellular morphology, proliferation capacity, growth rate and measurement of the content of telomere DNA. Giant senescent-like cells emerged shortly after cloning mouse PA317 and human HeLa cells expressing UUAGGG. The fraction of giant cells varied from 100% at the fifth population doubling (PD) in one culture to 2-6% at 50 PD in several other cultures. Giant cells were absent in all parental cells and clones expressing GGGAUU. The average cellular content of telomere DNA was independent of telomerase activity over 50 PD. The results indicate that expression of RNA complementary to the template region of hTR is sufficient to inhibit telomerase in vitro and in vivo, but that the effect of inhibition on individual cells is highly variable.

Animals↗

PubMed1998

Solution structures of the Huntington's disease DNA triplets, (CAG)n.

Highly polymorphic DNA triplet repeats, (CAG)n, are located inside the first exon of the Huntington's disease gene. Inordinate expansion of this repeat is correlated with the onset and progression of the disease. NMR spectroscopy, gel electrophoresis, digestion by single-strand specific P1 enzyme, and in vitro replication assay have been used to investigate the structural basis of (CAG)n expansion. Nondenaturing gel electrophoresis and 1D 1H NMR studies of (CAG)5 and (CAG)6 reveal the presence of hairpins and mismatched duplexes as the major and minor populations respectively. However, at high DNA concentrations (i.e., 1.0-2.0 mM that is typically required for 2D NMR experiments) both (CAG)5 and (CAG)6 exist predominantly in mismatched duplex forms. Mismatched duplex structures of (CAG)5 and (CAG)6 are useful, because they adequately model the stem of the biologically relevant hairpins formed by (CAG)n. We, therefore, performed detailed NMR spectroscopic studies on the duplexes of (CAG)5 and (CAG)6. We also studied a model duplex, (CGCAGCG)2 that contains the underlined building block of the duplex. This duplex shows the following structural characteristics: (i) all the nucleotides are in (C2'-endo, anti) conformations, (ii) mismatched A x A base pairs are flanked by two Watson-Crick G x C base pairs and (iii) A x A base pairs are stably stacked (and intra-helical) and are formed by a single N6-H--N1 hydrogen bond. The nature of A x A pairing is confirmed by temperature-dependent HMQC and HMQC-NOESY experiments on the [(CA*G)5]2 duplex where the adenines are 15N-labeled at N6. Temperature- and pH-dependent imino proton spectra, nondenaturing electrophoresis, and P1 digestion data demonstrate that under a wide range of solution conditions longer (CAG)n repeats (n> or =10) exist exclusively in hairpin conformation with two single-stranded loops. Finally, an in vitro replication assay with (CAG)8,21 inserts in the M13 single-stranded DNA templates shows a replication bypass for the (CAG)21 insert but not for the (CAG)8 insert in the template. This demonstrates that for a sufficiently long insert (n=21 in this case), a hairpin is formed by the (CAG)n even in presence of its complementary strand. This observation implies that the formation of hairpin by the (CAG)n may cause slippage during replication and thus may explain the observed length polymorphism.

DNA↗

PubMed1998

Hairpin induced slippage and hyper-methylation of the fragile X DNA triplets.

The fragile X triplet repeats, (GCC)n x (GGC)n are located at the 5' untranslated region of the FMR-1 gene. Inordinate repeat expansion and hyper-methylation of the CpG islands inside the repeat lead to the suppression of the FMR-1 gene and the subsequent onset and progression of the disease. Previously, we have shown that the (GCC)n strand of the fragile X repeat readily forms hairpin structures under physiological conditions (Chen et al., Proc. Natl. Acad. Sci. USA, 92:5199-5203, 1995: Mariappan et al., Nucl. Acid Res. 24:784-792, 1996). Here, we show by an in vitro assay that formation of the (GCC)n hairpins leads to slippage during replication. The slippage structure is a three-way junction with two Watson-Crick, (GCC)n x (GGC)n, arms and a third (GCC)n hairpin arm. Formation of such slippage structures during replication may explain the observed length polymorphism of the fragile X repeat. We have also studied the substrate efficiency of these three-way junctions toward the human methyltransferase. the enzyme that methylates the CpG sites in DNA. These methylation studies show that the slippage structures induced by the (GCC)n hairpins are 10-15 times more efficient substrates than either the corresponding Watson-Crick duplexes or the (GCC)n hairpins. We demonstrate by appropriate designs that the exceptional substrate efficiency of the three-way junction slippage structures is due to two factors: (i) the presence of the (GCC)n hairpin in which CpG sites are more accessible for methylation than the CpG sites in the Watson-Crick duplex and (ii) the ability of the (GCC)n hairpin in these three-way junctions to move along the Watson-Crick arms that facilitates conversion of low-affinity Watson-Crick CpG sites into high-affinity hairpin CpG sites. Therefore, we suggest that the formation of the (GCC)n hairpins during replication can explain both length polymorphism and hyper-methylation of the fragile X repeats.

Base Sequence↗

PubMed1998

Construction of an approximately 700-kb transcript map around the familial Mediterranean fever locus on human chromosome 16p13.3.

We used a combination of cDNA selection, exon amplification, and computational prediction from genomic sequence to isolate transcribed sequences from genomic DNA surrounding the familial Mediterranean fever (FMF) locus. Eighty-seven kb of genomic DNA around D16S3370, a marker showing a high degree of linkage disequilibrium with FMF, was sequenced to completion, and the sequence annotated. A transcript map reflecting the minimal number of genes encoded within the approximately 700 kb of genomic DNA surrounding the FMF locus was assembled. This map consists of 27 genes with discreet messages detectable on Northerns, in addition to three olfactory-receptor genes, a cluster of 18 tRNA genes, and two putative transcriptional units that have typical intron-exon splice junctions yet do not detect messages on Northerns. Four of the transcripts are identical to genes described previously, seven have been independently identified by the French FMF Consortium, and the others are novel. Six related zinc-finger genes, a cluster of tRNAs, and three olfactory receptors account for the majority of transcribed sequences isolated from a 315-kb FMF central region (between D16S468/D16S3070 and cosmid 377A12). Interspersed among them are several genes that may be important in inflammation. This transcript map not only has permitted the identification of the FMF gene (MEFV), but also has provided us an opportunity to probe the structural and functional features of this region of chromosome 16.

Amino Acid Sequence↗

PubMed1997

Cystosine-rich strands of the insulin minisatellite adopt hairpins with intercalated cytosine+.cytosine pairs.

Previously, we reported the high resolution NMR structure of the hairpin G-quartet structure formed by the G-rich strand of the insulin minisatellite of repeat sequence, (ACAG4TGTG4/TGTC4ACAC4) located upstream of the human insulin gene. Here, we report structural studies on the C-rich strand of this insulin minisatellite. First, we show by high resolution NMR that (C4TGTC4) forms a hairpin dimer with intercalated C+.C pairs (referred to as the hairpin i-motif); 340 NOE distance constraints uniquely define the nature of hairpin folding and the pattern of C+.C intercalation. Second, we show by one-dimensional NMR spectroscopy and molecular modeling studies that (C4TGTC4ACA4TGTC4) forms an intramolecularly folded hairpin with intercalated C+.C pairs. Third, we demonstrate by in vitro replication studies that several such hairpin i-motifs are present in long (C4TGTC4ACA)n (n>/=6) sequences, even in the presence of their complementary strands. Finally, we discuss structural and biological significance of the hairpin i-motifs formed by the C-rich strands of the insulin minisatellite.

Base Sequence↗

PubMed1997

Genomic structure and complete nucleotide sequence of the Batten disease gene, CLN3.

We recently cloned a cDNA for CLN3, the gene for juvenile-onset neuronal ceroid lipofuscinosis or Batten disease. To resolve the genomic organization we used a cosmid clone containing CLN3 to sequence the entire gene in addition to 1.1 kb 5' of the start of the published CLN3 cDNA and 0.3 kb 3' to the polyadenylation site. CLN3 is organized into at least 15 exons spanning 15 kb and ranging from 47 to 356 bp. The 14 introns vary from 80 to 4227 bp, and all exon/intron junction sequences conform to the GT/AG rule. Numerous repetitive Alu elements are present within the introns and 5'- and 3'-untranslated regions. The 5' region of the CLN3 gene contains several potential transcription regulatory elements but no consensus TATA-1 box was identified. CLN3 is homologous to 27 deposited human ESTs, and sequence comparisons suggest alternative splicing of the gene and the existence of transcribed sequences upstream to the start of the published CLN3 cDNA.

Base Sequence↗

PubMed1997

A radiation hybrid map of human chromosome 5 with integration of cytogenetic, genetic, and transcript maps.

One of the major goals of the human genome project is to establish a physical map of each human chromosome with a density of sequence-tagged site (STS) markers exceeding one every 100 kb. We report here the generation of a human chromosome 5-specific radiation hybrid (RH) map that includes 556 markers. Of these markers, 132 loci are ordered with a maximum likelihood ratio of >1000:1 compared with the next most likely order. An additional 113 loci were ordered relative to these backbone markers with a maximum likelihood ratio of >10:1 but <1000:1. Together, these 245 loci form an ordered framework map for the chromosome. Using this framework, >300 more markers were localized based on two-point analysis with the ordered set. On average, there are 50 markers in common with the RH map presented here and other chromosome 5 maps included in the current whole genome cytogenetic, genetic, and physical maps. The accuracy of all the maps is evident in that there are no more than two discrepancies between any one of them and these data. All of the maps encompassing chromosome 5 complement each other providing excellent STS coverage with >2200 loci combined. The chromosome 5-specific RH map contains 20% of these independent loci. In addition, our RH map contains STSs derived from clones suitable for fluorescent in situ hybridization, allowing alignment to the cytogenetic map. Together, these maps will assist in the assembly of sequence-ready contigs and will aid in the identification of disease loci on chromosome 5 by positional cloning and positional candidate approaches.

Centromere↗

PubMed1997

Measurement of telomeric DNA content in human tissues.

Telomeres, nucleoprotein complexes at the ends of eukaryotic chromosomes, are 10-12 kbp in length in somatic cells, but as small as 1-2 kbp in rapidly growing cancer cells. Southern blot analysis is currently the standard method for the measurement of telomere length. However, accurate determinations are not possible when DNA is broken or scant. To avoid these problems, a slot blot assay that quantitates the relative content, instead of length, of telomere DNA was developed. The relative contents of telomere DNA determined by this slot blot assay were directly proportional to the relative lengths of telomere DNA determined in parallel by Southern blot analysis. Relative telomere DNA content could be measured in samples containing as little as 15 ng of total DNA. Relative telomere DNA content, but not length, also was unaffected by breakage of DNA into fragments 1 kbp or less in length.

Base Composition↗

PubMed1996

Structure-function correlations of the insulin-linked polymorphic region.

The insulin minisatellite of the insulin-linked polymorphic region (ILPR), a 14 base-pairs long tandem repeat of: 5'-ACAGGGGTGTGGGG-3' 3'-TGTCCCCACACCCC-5', is located 363 base-pairs upstream of the human insulin gene. A locus for insulin-dependent diabetes mellitus (IDDM) has been mapped to the ILPR. It has been shown that the ILPR is polymorphic in length and this length polymorphism is also related to the transcriptional activity of the insulin gene and the susceptibility to IDDM. Here, we attempt to decipher the role of the ILPR structure in length polymorphism and transcriptional regulation. We show by gel electrophoresis, circular dichroism (CD) and one and two-dimensional nuclear magnetic resonance spectroscopy (1D/2D NMR) that the G-rich strand of the ILPR adopts an intramolecularly folded hairpin G-quartet structure. A detailed analysis of 1D/2D NMR data of d(G4TGTG4) and d(G4TGTG4ACAG4TGTG4) enables us to define the nature of chainfolding, the stacking interaction of the G-tetrads in the stem, and the interactions of the bases in the loops. d(G4TGTG4ACAG4TGTG4) happens to be the smallest unit of the G-rich strand that can form the intramolecular hairpin G-quartet structure. For long ILPR sequences, several such hairpin G-quartet structures can be linked in space. Indeed, by an in vitro replication assay, we show the presence of such multiple hairpin G-quartet structures for the G-rich strand of the ILPR of repeat length 6. This observation suggests that the formation of multiple hairpin G-quartets may explain slippage during replication and the observed length polymorphism. From our high resolution structure, we are able to identify a set of interactions that are critical for the structure and stability of the hairpin G-quartet. Single or double mutations in the ILPR that destabilize these interactions also lower the transcriptional activity of the insulin gene. Therefore, the hairpin G-quartet structure of the ILPR has a direct correlation with the transcriptional activity of the human insulin gene.

DNA↗