HumanDCTN1: Genomic Structure and Evaluation as a Candidate for Alström Syndrome

The human dynactin 1 gene (DCTN1) is positioned on chromosome 2p13, the candidate region for various diseases including Alström syndrome, limb-girdle muscle dystrophy, and Miyoshi myopathy. Here, we report the exon–intron structure ofDCTN1along with characterization of the 5′ upstream sequence and alternative splice variants previously identified by Tokitoet al.(1996),Mol. Biol. Cell7: 1167–1180). Knowledge of the genomic structure ofDCTN1allowed us to design intronic primers necessary for analyzing mutations in families segregating for diseases linked to this gene. These primers were tested on a French Acadian kindred segregating for Alström syndrome. No mutations were observed within the coding region ofDCTN1in this family. However, the intronic primers should allow for the rapid amplification of the coding region for mutational analysis of additional Alström families and other diseases tightly linked to theDCTN1locus on chromosome 2p13.     

Low-Molecular-Weight, Calcium-Dependent Phospholipase A2Genes Are Linked and Map to Homologous Chromosome Regions in Mouse and Human

The Group IIA phospholipase gene (PLA2G2A) protein coding regions exhibit significant homology with recently described Group IIC (PLA2G2C) and Group V (PLA2GV) genes. All three genes are present in many mammalian species and are expressed in a tissue-specific pattern. Here, we demonstrate in human that they are tightly linked and map to chromosome 1p34–p36.1. We also show that the homologous mouse loci are tightly linked (no observed recombination) on the distal part of chromosome 4, a region exhibiting synteny with human 1p34–p36. Unlike its rodent counterpart, humanPLA2G2Cappears to be a nonfunctional pseudogene.     

Linkage and physical mapping of prolactin to porcine chromosome 7

Comparative mapping studies between human and pig have shown that there is conserved synteny between human chromosome 6 and pig chromosomes 1 and 7, but some gene locations are not well established. Prolactin ( PRL ), an anterior pituitary hormone, has been mapped to human chromosome 6, and has tentatively mapped to pig chromosome 7 using Southern-RFLP analysis with a limited number of meioses. To confirm the assignment of prolactin to porcine chromosome 7 by physical and linkage analysis, pig cDNA and human genomic DNA sequences were used to design pig-specific PCR primers. The primers amplified a fragment of ≈2·8 kb. Two polymorphic restriction sites were identified within this fragment with the restriction endonuclease Bst UI. Prolactin was significantly linked to six markers on the published PiGMaP map of pig chromosome 7. Prolactin was physically mapped using a pig × rodent somatic cell hybrid panel. An analysis of these data placed PRL on pig 7p1·1–p1·2 with 100% concordance and was in complete agreement with the linkage data. Both mapping techniques placed PRL in a conserved order with the loci in the syntenic region of human chromosome 6.     

Linkage of the apo CIII microsatellite with isolated low high-density lipoprotein cholesterol

To evaluate whether a highly polymorphic mic-rosatellite region within intron 3 of the apolipoprotein (apo) CIII gene is linked to the isolated low HDL-C phenotype, we studied eight unrelated probands (mean HDL-C=10 ±5mg/dl) and 157 biological family members. After PCR amplification of genomic DNA and denaturing polyacrylamide gel electrophoresis, 26 alleles were identified in this microsatellite including 9 alleles heretofore unreported. Quantitative sib-pair linkage analysis demonstrated strong evidence of linkage between the isolated low HDL-C phenotype and the apo CIII microsatellite region (P=0.007). The microsatellite was also linked to apo AI (P=0.001), the primary apolipoprotein of HDL-C. Therefore, this highly polymorphic microsatellite region is a potentially important marker in the genetic evaluation of the isolated low HDL-C phenotype. Received: 25 October 1996 / Accepted: 29 October 1997     

Molecular mapping of a nuclear male-sterility gene in sunflower (Helianthus annuus L.) using TRAP and SSR markers

A nuclear male-sterile mutant, NMS 360, induced by streptomycin from an inbred maintainer line HA 89, possesses a single recessive gene, ms9, controlling male sterility. The present study identified DNA markers linked to the ms9 gene in an F₂ population derived from the cross of NMS 360 × RHA 271 and maps the ms9 gene to an existing sunflower SSR linkage map. Bulked segregant analysis was performed using the target region amplification polymorphism (TRAP) marker technique and the simple sequence repeats (SSR) technique. From 444 primer combinations, six TRAP markers linked with the ms9 gene were amplified. Two markers, Ts4p03-202 and Tt3p09-529, cosegregated with the ms9 gene. The other four markers, To3d14-310, Tt3p17-390, Ts4p23-300, and Tt3p09-531, linked with ms9 at a distance of 1.2, 3.7, 10.3, and 22.3 cM, respectively. Thirty SSR primers from 17 linkage groups of a PHA × PHB cultivated sunflower linkage map were screened among the two parents and the F₂ population. SSR primer ORS 705 of linkage group 10 was tightly linked to ms9 at a distance of 1.2 cM. The ms9 gene was subsequently mapped to linkage group 10 of the public sunflower SSR linkage map. The markers that were tightly linked with the ms9 gene will be useful in marker-assisted selection of male-sterile plants among segregating populations, and will facilitate the isolation of the ms9 gene by map-based cloning.     

猪Pit-1基因第三内含子序列的克隆测序

根据不同物种间同一基因核苷酸序列的保守性及相似性的特点 ,在人和鼠的Pit 1基因第三外显子上设计上游引物 ,而将下游引物设计在猪Pit 1基因第四外显子上。利用聚合酶链式反应 (PCR)技术 ,扩增出猪Pit 1基因第三内含子序列 ,并经由酶切及序列同源性比对确认该序列即为猪Pit 1基因第三内含子序列。此序列的确定为下一步进行遗传变异分析的研究奠定了基础     

Assignment of the gene for porcine insulin-like growth factor 1 (IGF1) to chromosome 5 by linkage mapping

Investigation of published sequence data from the porcine insulin-like growth factor 1 (IGF1) gene, resulted in the detection of a microsatellite in the first intron of the gene. Polymerase chain reaction (PCR) primers flanking the (CA)₁₉ repeat were constructed. Polymorphism and Mendelian segregation were documented in a three-generation pedigree and allele frequencies were determined in 74 unrelated animals from four different breeds. Seven alleles were encountered. Linkage analysis was performed in a large pedigree established for gene mapping. Linkage between the IGF1 microsatellite and an anonymous microsatellite marker, S0005, was detected. Furthermore, IGF1 and S0005 was found to be linked to the porcine submaxillary gland mucin (MUC) gene, previously assigned to chromosome 5. The results presented here extend the linkage group on pig chromosome 5 and are in accordance with conserved synteny between human chromosome 12, cattle chromosome 5, mouse chromosome 10 and pig chromosome 5.     

Chromosomal assignment of the human genes coding for the major proteins of the desmosome junction, desmoglein DGI (DSG), desmocollins DGII/III (DSC), desmoplakins DPI/II (DSP), and plakoglobin DPIII (JUP)

We have established PCR assays for the genes coding for the major proteins of the desmosome type of cell junction, the desmosomal cadherins DGI (desmoglein) and DGII/III (desmocollins), and the plaque proteins DPI/II (desmoplakin) and DPIII (plakoglobin) and used them to test human-mouse and human-rat somatic cell hybrids with different contents of human chromosomes. From these data we were able to assign DGI to chromosome 18 (DSG), DGII/III to chromosome 9p (DSC), DPI/II to chromosome 6p21-ter(DSP), and DPIII to chromosome 7 (JUP).     

Map order and linkage distances of molecular markers close to the supernodulation ( nts-1 ) locus of soybean

The molecular characteristics of markers in the chromosome region surrounding the supernodulation gene (nts-1) of soybean (Glycine max L. Merr.) were investigated in 187 F₂ plants from a cross of G. max cv. Bragg (nts) and G. soja PI468.397 (wild-type nodulation). RFLP marker pUTG-132a, linked tightly (0.7±0.5 cM) to nts-1, was converted to a PCR marker. The polymorphism resides within a 1.72 kb PstI fragment and consists of an 832 bp insertion in G. max relative to the wild progenitor G. soja. The insertion is flanked by a 35 bp direct duplication that was found only once in G. soja. Data suggest that the pUTG-132a sequence exists only once in the genome, which is compatible with the recessive nature of nts-1. Accordingly, pUTG-132a is a valuable marker for map-based cloning. Another RFLP marker, pA-381, was mapped 4.8 cM distal to nts-1. Marker order, established by Maximum Likelihood Analysis, placed nts-1 between pUTG-132a and pA-381. To generate additional molecular markers, a segregating F₂ population was analysed using bulked segregant analysis (BSA) and single oligonucleotide primer-based PCR (DNA amplification fingerprinting; DAF). PCR marker pcr5-4L was mapped to soybean linkage group H and sequenced. The data revealed (i) recombination events and marker order in the nts-1 region; (ii) the molecular nature and cause of polymorphisms in linked molecular markers; (iii) a low density of polymorphisms around nts-1, and (iv) diploidy of the distal region of linkage group H of soybean. Received: 18 January 1996 / Accepted: 9 October 1996     

Isolation, mapping, and characterization of two cDNA clones expressed in the cerebellum.

In an effort to characterize genes expressed in the cerebellum, we have isolated two cDNA clones, H11B (D16S286) and 507 (D5S344), that hybridized to a cerebellar cDNA probe. Using a panel of human-rodent somatic cell hybrids, cDNA clone H11B was mapped to human chromosome 16, and clone 507 was mapped to human chromosome 5. TaqI RFLPs were identified with both clones and were used for linkage analysis in the CEPH families. D16S286 was tightly linked to several markers near chromosome 16p13, and D5S344 was tightly linked to several markers on chromosome 5q. Sequence tagged sites or expressed sequence tags were generated from the 3' untranslated regions of both cDNA clones.     

Fine mapping of the 2p11 dyslexia locus and exclusion of TACR1 as a candidate gene

Developmental dyslexia, or reading disability, is a multigenic complex disease for which at least five loci, i.e. DYX1–3 and DYX5–6, have been clearly identified from the human genome. To date, DYX1C1 is the only dyslexia candidate gene cloned. We have previously reported linkage to 2p11 and 7q32 in 11 Finnish pedigrees. Here, we report the fine mapping of the approximately 40-cM linked region from chromosome 2 as we increased marker density to one per 1.8 cM. Linkage was supported with the highest NPL score of 3.0 (P=0.001) for marker D2S2216. Association analysis using the six pedigrees showing linkage pointed to marker D2S286/rs3220265 (P value <0.001) in the near vicinity of D2S2216. We went on to further characterise this ~15-cM candidate region (D2S2110-D2S2181) by adding six SNPs covering ~670 kb centred at D2S286/rs3220265. A haplotype pattern could no longer be observed in this region, which was therefore excluded from the candidate area. This also excluded the TACR1 (tachykinin receptor 1) gene, located at marker D2S286. The dyslexia candidate region on 2p11 is, therefore, now limited to the chromosomal area D2S2116-D2S2181, which is ~12 Mbp of human sequence and is at a distinct location from the previously reported DYX3 locus, raising the possibility of two distinct loci on chromosome 2p.H. Anthoni and P. Onkamo contributed equally to this work     

New regional localisations for HAGH and PGP on human chromosome 16

Summary The chromosomal locations for the electrophoretic markers hydroxyacyl glutathione hydrolase (HAGH) and phosphoglycolate phosphatase (PGP) were examined using a human-mouse hybrid panel of chromosome 16. The assignment for HAGH was confirmed to chromosome 16 using a cell line with chromosome 16 as the only human chromosome. Both HAGH and PGP were present only in cell lines containing human 16p13. This localisation for PGP indirectly places the tightly linked genes for the alpha-globin cluster and adult polycystic kidney disease on 16p13.     

Mapping the human acetylcholinesterase gene to chromosome 7q22 by fluorescent in situ hybridization coupled with selective PCR amplification from a somatic hybrid cell panel and chromosome-sorted DNA libraries

To establish the chromosomal location of the human ACHE gene encoding the acetylcholine hydrolyzing enzyme acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, E.C. 3.1.1.7), a human-specific polymerase chain reaction (PCR) procedure that supports the selective amplification of ACHE DNA fragments from human genomic DNA was employed with 19 human-hamster somatic cell hybrids carrying one or more human chromosomes. Informative ACHE-specific PCR fragments were produced from two cell lines, both of which include human chromosome 7, but not with DNA from 17 cell hybrids carrying various combinations of all human chromosomes other than 7. Fluorescent in situ hybridization of biotinylated ACHE DNA with metaphase chromosomes from human peripheral blood lymphocytes revealed prominent labeling on the 7q22 position. Therefore, further tests were performed to confirm the chromosome 7 location. DNA samples from the two cell lines including chromosome 7 and the ACHE gene were positive with PCR primers informative for the human cystic fibrosis CFTR gene, known to reside at the 7q31.1 position, but negative for the ACHE-related butyrylcholinesterase (BCHE, acylcholine acylhydrolase, E.C. 3.1.1.8) gene, mapped at the 3q26-ter position, confirming that these lines contain chromosome 7 but not chromosome 3. In contrast, three other cell lines including chromosome 3, but not 7, were BCHE-positive and ACHE-negative. In addition, genomic DNA from a sorted chromosome 7 library supported the production of ACHE- but not BCHE-specific PCR products, whereas with DNA from a sorted chromosome 3 library, the BCHE but not the ACHE fragment was amplified. These findings assign the human ACHE gene to a single locus on chromosome 7q22 and should assist in establishing linkage between the in vivo amplification of the ACHE gene in ovarian tumors and leukemias and the phenomenon of tumor-related breakage in the long arm of chromosome 7.     

Phosphoenolpyruvate Carboxykinase (GTP): Characterization of the Human PCK1 Gene and Localization Distal to MODY on Chromosome 20

The human PCK1 gene encoding phosphoenolpyruvate carboxykinase (GTP) (PEPCK) was isolated and sequenced. There is 91% amino acid sequence identity (567/622 residues) between the human and the rat proteins, with conservation of intron/exon borders. A polymorphic dinucleotide microsatellite with the structure (CA)₁₆(TA)₅(CA) was identified in the 3′ untranslated region of the cloned human PCK1 gene. This highly informative genetic marker has an estimated PIC value of 0.79 and heterozygosity of 0.81. Analysis of the RW pedigree demonstrated recombination between PCK1 and the MODY gene on chromosome 20. Multipoint linkage analysis of the reference pedigrees of the Centre d'Etude du Polymorphisme Humain localized PCK1 on the genetic map of chromosome 20 at a position distal to markers that are closely linked to MODY. PCK1 is part of a conserved linkage group on mouse Chromosome 2 with identical gene order but expanded length in the human genome.     

Sequence, organization and expression of the core histone genes of Aspergillus nidulans

Summary The core histone gene family ofAspergillus nidulans was characterized. The H2A, H2B and H3 genes are unique in theA. nidulans genome. In contrast there are two H4 genes, H4.1 and H4.2. As previously reported for the H2A gene (May and Morris 1987) introns also interrupt the other core histone genes. The H2B gene, like the H2A gene, is interrupted by three introns, the H3 and H4.1 gene are each interrupted by two introns and the H4.2 gene contains one intron. The position of the single intron in H4.2 is the same as that the first intron of the H4.1 gene. The H2A and H2B genes are arranged as a gene pair separated by approximately 600 by and are divergently transcribed. The H3 and H4.1 genes are similarly arranged and are separated by approximately 800 bp. The H4.2 gene is not closely linked to either the H2A-H2B or H3-H4.1 gene pairs. Using pulse field gel electrophoresis an electrophoretic karyotype was established forA. nidulans. This karyotype was used to assign the H3–H4.1 gene pair and the H4.2 gene to linkage group VIII and the H2A–H2B gene pair to either linkage group III or VI. The abundance of each of the histone messenger RNAs was determined to be cell cycle regulated but the abundance of the H4.2 mRNA appears to be regulated differently from the others.     

New polymorphisms and markers in the HLA class I region: relevance to hereditary hemochromatosis (HFE)

Hereditary hemochromatosis (HFE) is an inherited disorder whose gene lies in the proximity of the histocompatability antigen (HLA) class I region, on 6p21.3. Despite efforts in refining the HFE region, a number of informative DNA markers, linked to the disease locus and amenable to use in an assay based on the polymerase chain reaction (PCR) is available. The gene content of this region is high, and the HFE gene has not so far been identified. We have used a strategy based on PCR protocols potentially able to detect both polymorphisms and expressed sequences. This approach has been applied to a 700-kb stretch (approximately) of DNA corresponding to the insert of a Centre d'Etude du Polymorphisme Humain yeast artificial chromosome (225 B1) of the possible candidate region. Five new polymorphisms have been detected among 20 specific fragments isolated. Four of them are tightly linked to the HFE locus. Because of the strong linkage disequilibrium with the disease demonstrated by these markers, they could represent starting points for the identification and characterization of the HFE gene. The remaining non-polymorphic fragments, being amplifiable and in most cases linked to NotI sites, may be useful starting points for the generation of a genomic contig of band 6p21.3 and for gene identification.     

Genetic mapping of the human tryptophan hydroxylase gene on chromosome 11, using an intronic conformational polymorphism.

The identification of polymorphic alleles at loci coding for functional genes is crucial for genetic association and linkage studies. Since the tryptophan hydroxylase (TPH) gene codes for the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, it would be advantageous to identify a polymorphism in this gene. By examining introns of the human TPH gene by PCR amplification and analysis by the single-strand conformational polymorphism (SSCP) technique, an SSCP was revealed with two alleles that occur with frequencies of .40 and .60 in unrelated Caucasians. DNAs from 24 informative CEPH families were typed for the TPH intron polymorphism and analyzed with respect to 10 linked markers on chromosome 11, between p13 and p15, with the result that TPH was placed between D11S151 and D11S134. This region contains loci for several important genes, including those for Beckwith-Wiedemann syndrome and tyrosine hydroxylase.     

Isolation of monochromosomal hybrids for mouse Chromosomes 3, 6, 10, 12, 14, and 18

Mouse/human somatic cell hybrids constitute a valuable resource for both genetic and physical mapping. In this report, we describe the production and characterization of a series of six monochromosomal hybrids generated by fusion of murine microcells with intact human recipient cells. The presence of each mouse chromosome was characterized by PCR analysis and the integrity of the mouse chromosome retained in the hybrids confirmed by fluorescence in situ hybridization (FISH) analysis. Received: 22 August 1996 / Accepted: 19 September 1996     

Linkage disequilibrium between the FES, D15S127, and BLM loci in Ashkenazi Jews with Bloom syndrome.

Bloom syndrome (BS) is more common in the Ashkenazi Jewish than in any other population. Approximately 1 in 110 Ashkenazi Jews carries blm, the BS mutation. The locus mutated in BS, BLM, maps to chromosome subband 15q26.1, tightly linked to the proto-oncogene FES. We have investigated the basis for the increased frequency of blm in the Ashkenazim by genotyping polymorphic microsatellite loci tightly linked to BLM in affected and unaffected individuals from Ashkenazi Jewish and non-Ashkenazi populations. A striking association of the C3 allele at FES with blm (delta = .422; p = 5.52 x 10(-7)) and of the 145-bp and 147-bp alleles at D15S127 with blm (delta = .392 and delta = .483, respectively; p = 2.8 x 10(-5) and p = 5.4 x 10(-7), respectively) was detected in Ashkenazi Jews with BS. This linkage disequilibrium constitutes strong support for a founder-effect hypothesis: the chromosome in the hypothetical founder who carried blm also carried the C3 allele at FES and either the 145-bp or the 147-bp allele at D15S127.